• View in gallery

    Representative photomicrographs indicating results of immunohistochemical staining with PU17 polyclonal antibody to detect FRs in dogs with multicentric lymphoma. A—Section of normal canine kidney tissue used as a positive control specimen. There is intense immunoreactivity to PU17 on the apical surface of proximal tubular epithelial cells, which corresponds to the expected location of FRa. B—Section of a lymph node biopsy specimen stained with PU17. There are scattered cells with diffuse cytoplasmic immunoreactivity to PU17; these may represent tumor-associated macrophages expressing FRb. C—Section of a negative control sample stained with nonimmune serum. Bar = 100 μm.

  • View in gallery

    Right lateral scintigraphic images of 2 dogs with DLBCL that had (A) and did not have (B) uptake of 99mTc-EC20 during nuclear scintigraphy. In panel A, notice the uptake of 99mTc-EC20 in the mandibular (asterisk), medial retropharyngeal (dagger), and superficial cervical (double dagger) lymph nodes.

  • View in gallery

    Right lateral scintigraphic images of a dog with stage 5 (according to World Health Organization criteria) DLBCL. The dog was treated with CHOP and had complete remission for 3 months before cancer relapse occurred. A—There is nonspecific uptake of 99mTc-EC20 by the liver (asterisk) and kidneys (dagger), with accumulation of excreted radioisotope in the urinary bladder (double dagger). B—Shielding of the abdominal viscera (large black circle) allows visualization of 99mTc-EC20 uptake in the mandibular (asterisk) and superficial cervical (dagger) lymph nodes, lungs and tracheobronchial lymph nodes (double dagger), and subcutaneous fat along the dorsum (number sign). C—Repeated nuclear scintigraphy at the time of cancer relapse reveals a lack of tumoral uptake of 99mTc-EC20.

  • 1. Priester WA, McKay FW. The occurrence of tumors in domestic animals. Natl Cancer Inst Monogr 1980; 54: 1210.

  • 2. Valli VE, San Myint M, Barthel A, et al. Classification of canine malignant lymphomas according to the World Health Organization criteria. Vet Pathol 2011; 48: 198211.

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  • 3. Vezzali E, Parodi AL, Marcato PS, et al. Histological classification of 171 cases of canine and feline non-Hodgkin lymphoma according to the WHO. Vet Comp Oncol 2009; 8: 3849.

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  • 9. Chun R, Garrett LD, Vail DM. Evaluation of a high-dose chemotherapy protocol with no maintenance for dogs with lymphoma. J Vet Intern Med 2000; 14: 120124.

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  • 10. Low PS, Henne WA, Doorneweerd DD. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res 2008; 41: 120129.

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  • 11. Salazar MD, Ratnam M. The folate receptor: what does it promise in tissue-targeted therapeutics? Cancer Metastasis Rev 2007; 26: 141152.

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  • 13. Shmeeda H, Mak L, Tzemach D, et al. Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors. Mol Cancer Ther 2006; 5: 818824.

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  • 14. Elnakat H, Ratnam M. Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Adv Drug Deliv Rev 2004; 56: 10671084.

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  • 15. Leamon CP, Parker MA, Vlahov IR, et al. Synthesis and biological evaluation of EC20: a new folate-derived 99mTc-based radiopharmaceutical. Bioconjugate Chem 2002; 13: 12001210.

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  • 16. Reddy JA, Dorton R, Westrick E, et al. Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate. Cancer Res 2007; 67: 44344442.

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  • 17. Lorusso PM, Edelman JM, Bever SL, et al. Phase I study of folate conjugate EC145 (Vintafolide) in patients with refractory solid tumors. J Clin Oncol 2012; 30: 40114016.

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  • 18. Dhawan D, Ramos-Vara JA, Naughton JF, et al. Targeting folate receptors to treat invasive urinary bladder cancer. Cancer Res 2012; 73: 875884.

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  • 19. Vail DM, Michels GM, Khanna C, et al. Response evaluation criteria for peripheral nodal lymphoma in dogs (v1.0)—a veterinary cooperative oncology group (VCOG) consensus document. Vet Comp Oncol 2009; 8: 2837.

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  • 20. Skipper HE, Schabel FM, Mellett LB, et al. Implications of biochemical, cytokinetic, pharmacologic, and toxicologic relationships in the design of optimal therapeutic schedules. Cancer Chemother Rep 1970; 54: 431450.

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  • 21. Veterinary Co-operative Oncology Group. Veterinary Co-operative Oncology Group—common terminology criteria for adverse events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.0. Vet Comp Oncol 2004; 2: 194213.

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Assessment of folate receptor expression and folate uptake in multicentric lymphomas in dogs

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  • 1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 2 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 3 Endocyte Inc, 3000 Kent Ave, Ste A1-100, West Lafayette, IN 47906.
  • | 4 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 5 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 6 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 7 Department of Chemistry, College of Science, Purdue University, West Lafayette, IN 47907
  • | 8 Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907.
  • | 9 Endocyte Inc, 3000 Kent Ave, Ste A1-100, West Lafayette, IN 47906.
  • | 10 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 11 Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907.

Abstract

Objective—To determine expression of folate receptors (FRs) and folate uptake in multicentric lymphomas in dogs.

Sample—10 dogs with histopathologically confirmed multicentric lymphoma and 20 archival lymph node biopsy specimens from dogs with multicentric lymphoma.

Procedures—Multicentric lymphomas in 10 dogs were prospectively evaluated for FR expression by use of immunohistochemical analysis and for in vivo folate uptake by use of nuclear scintigraphy. Dogs with FR-expressing tumors were eligible for FR-targeted chemotherapy. Twenty archival lymphoma biopsy specimens were also evaluated with immunohistochemical analysis.

Results—FRs were not detected with immunohistochemical analysis in lymph node samples obtained from the 10 dogs or in archival biopsy specimens. However, nuclear scintigraphy revealed uptake of radioactive tracer in 6 of 10 dogs. Five of these 6 dogs were treated with an FR-targeted chemotherapeutic agent; results of treatment were complete remission in 1 dog, stable disease in 2 dogs, and progressive disease in 2 dogs. Treatment-related toxicoses generally were mild.

Conclusions and Clinical Relevance—This study provided strong evidence for folate uptake in a substantial portion of multicentric lymphomas of dogs and indicated the antitumor activity of FR-targeted chemotherapeutics for these cancers. Use of FR-targeted chemotherapeutics may be promising for the treatment of FR-expressing multicentric lymphomas in dogs. Further studies are needed to determine reasons for lack of immunoreactivity to currently identified anti-FR antibodies and to develop improved methods for detecting FRs in lymphomas of dogs.

Abstract

Objective—To determine expression of folate receptors (FRs) and folate uptake in multicentric lymphomas in dogs.

Sample—10 dogs with histopathologically confirmed multicentric lymphoma and 20 archival lymph node biopsy specimens from dogs with multicentric lymphoma.

Procedures—Multicentric lymphomas in 10 dogs were prospectively evaluated for FR expression by use of immunohistochemical analysis and for in vivo folate uptake by use of nuclear scintigraphy. Dogs with FR-expressing tumors were eligible for FR-targeted chemotherapy. Twenty archival lymphoma biopsy specimens were also evaluated with immunohistochemical analysis.

Results—FRs were not detected with immunohistochemical analysis in lymph node samples obtained from the 10 dogs or in archival biopsy specimens. However, nuclear scintigraphy revealed uptake of radioactive tracer in 6 of 10 dogs. Five of these 6 dogs were treated with an FR-targeted chemotherapeutic agent; results of treatment were complete remission in 1 dog, stable disease in 2 dogs, and progressive disease in 2 dogs. Treatment-related toxicoses generally were mild.

Conclusions and Clinical Relevance—This study provided strong evidence for folate uptake in a substantial portion of multicentric lymphomas of dogs and indicated the antitumor activity of FR-targeted chemotherapeutics for these cancers. Use of FR-targeted chemotherapeutics may be promising for the treatment of FR-expressing multicentric lymphomas in dogs. Further studies are needed to determine reasons for lack of immunoreactivity to currently identified anti-FR antibodies and to develop improved methods for detecting FRs in lymphomas of dogs.

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.

Materials and Methods

Sample—The study included dogs with multicentric lymphoma and archival lymph node biopsy specimens. Ten dogs with multicentric lymphoma treated at the Purdue University Veterinary Teaching Hospital were enrolled. Written informed consent was obtained from all owners prior to enrollment of each dog. All procedures for these dogs were performed with the approval of the Purdue Animal Care and Use Committee.

Dogs were eligible for enrollment in the study if they had cytologically or histopathologically diagnosed multicentric lymphoma. Surgical biopsy of lymph nodes was performed in dogs with only a cytologic diagnosis of lymphoma; these biopsy specimens were used for histopathologic confirmation and lymphoma subtyping in accordance with World Health Organization criteria.2 Dogs with untreated lymphoma, lymphoma that had relapsed following initial complete remission (ie, relapsed lymphoma), or lymphoma that was progressing despite ongoing treatment (ie, resistant lymphoma) were eligible for the study. Dogs were ineligible if they had lymphoma of a nonmulticentric (ie, primarily extranodal) distribution or if they had an expected survival time < 6 weeks (with or without treatment). It was required that all dogs have lymphoma with involvement of peripheral lymph nodes, with affected nodes measuring at least 10 mm in the longest dimension.19 Lymph node biopsy specimens were prospectively collected by use of incisional wedge biopsy or surgical extirpation of an affected lymph node from the 10 dogs, except for 1 prospectively collected sample that was obtained via needle core biopsy. The dog in which the needle core biopsy was performed had resistant lymphoma; a specimen had been obtained 4 months previously via incisional wedge biopsy for diagnostic evaluation and subtyping in accordance with World Health Organization criteria.2

Twenty archival lymph node biopsy specimens from dogs with multicentric lymphoma were retrieved from the Indiana Animal Disease Diagnostic Laboratory. These archival specimens included 10 DLBCLs, 1 B-lymphoblastic lymphoma, and 9 with PTCL-NOS. All 20 archival lymph node biopsy specimens had been obtained by use of incisional wedge biopsy or surgical extirpation of an affected lymph node.

Immunohistochemical analysis—Immunohistochemical analysis was performed on formalin-fixed tissue sections as described elsewhere.18 Briefly, 5- mm-thick sections were cut from paraffin-embedded tissues and placed on glass slides. Sections were deparaffinized in xylene and rehydrated in descending serial concentrations of alcohol. A modified citrate buffer solutionb was used for antigen retrieval in accordance with the manufacturer's instructions. Tissue sections were then immersed in 3% hydrogen peroxide to block endogenous peroxidase activity and blocked with a purified casein-blocking reagent.c This was followed by incubation with primary antibody (ie, PU17)d for 2 hours at room temperature (approx 21°C); PU17 is a rabbit polyclonal antibody raised against the bovine milk folate-binding protein, which shares 90% sequence homology with human FRα. In addition, PU17 has been validated for the immunohistochemical detection of FRs in human and canine tissues. Paired negative control slides from each biopsy specimen were stained with negative control serum.e Canine kidney tissues served as a positive control sample. The FR immunoreactive complexes were detected with a horseradish peroxidase–based antibody detection system.f Immune complexes were developed with 3, 3′-diaminobenzidine substrateg and counterstained with hematoxylin-1.h All slides were reviewed separately by 2 board-certified veterinary pathologists (MAM and JARV), and a consensus interpretation was reported.

Nuclear scintigraphy—All 10 dogs were subjected to nuclear scintigraphy with a folate-technetium conjugate,i as described elsewhere.18 Briefly, 5 mCi of 99mTc was added to a solution of a folate-containing peptidej and injected IV into each dog 2 hours before image acquisition. All dogs were anesthetized for image acquisition. Full-body static images were obtained with dogs in dorsoventral, ventrodorsal, and right and left lateral recumbencies over a period of 90 s/view by use of a single-head gamma camera.k All images were reviewed by a board-certified veterinary radiologist (JFN).

FR-targeted chemotherapeutic treatment—Dogs with multicentric lymphoma in which FRs were detected with immunohistochemical analysis or nuclear scintigraphy were eligible for FR-targeted chemotherapeutic treatment. The agent used was EC0905,l a folate-vinblastine conjugate drug that is 28.4% vinblastine by weight. Treated dogs received EC0905 at a dose of 0.25 mg/kg, IV, once weekly; this dose has previously been found to be the maximally tolerated dose in dogs.18 None of the dogs that received EC0905 were receiving other treatments for lymphoma during the time they received the EC0905, except for 1 dog that was receiving prednisone (approx 2.7 mg/kg, PO, q 24 h) for 3 weeks prior to enrollment in the study. The lymphoma of that dog had progressed despite the prednisone treatment (ie, resistant lymphoma), and the prednisone dose was gradually tapered during the first 3 weeks that the dog received EC0905.

Response to EC0905 treatment was classified as complete remission, partial remission, stable disease, or progressive disease on the basis of previously defined criteria.19 An objective response to chemotherapy was defined as a best response of complete remission or partial remission that lasted for at least 3 weeks, whereas dogs with complete remission or partial remission lasting < 3 weeks or with a best response of stable disease or progressive disease were considered nonresponders. Dogs with complete remission received a maximum of 6 weekly doses of EC0905, and then treatment was discontinued and the dogs monitored for cancer relapse. This treatment regimen was developed on the basis of cytokinetic models that suggested a minimum of 6 cycles of chemotherapy would be necessary to result in a reasonable chance for cure of chemoresponsive malignancies.20 Dogs with partial remission or stable disease were treated until complete remission or progressive disease was detected. Treatment with EC0905 was discontinued in dogs with progressive disease, but these dogs were provided rescue chemotherapy at the discretion of the owner and attending veterinarian.

Treatment-related toxicoses were assessed at weekly intervals via physical examinations, owner observations, CBCs, and serum biochemical profiles, as previously described for dogs receiving EC0905 treatment.18 Treatment-related toxicoses were scored in accordance with the Veterinary Cooperative Oncology Group criteria.21 Dose reductions were 10% for grade 1 or 2 toxicoses and 20% for toxicoses of ≥ grade 3.

Results

Dogs—Dogs with multicentric lymphoma enrolled in the study included 8 dogs with DLBCL, 1 dog with PTCL-NOS, and 1 dog with nodal marginal zone lymphoma. There were 6 castrated male dogs and 4 spayed female dogs. Median age was 7.9 years (range, 3.0 to 12.3 years), and median body weight was 20.8 kg (range, 7.7 to 37.0 kg). Five dogs had not received treatment for lymphoma, and 5 dogs had resistant lymphoma. Prior treatments in the dogs with resistant lymphoma included prednisone in 3 dogs, prednisone and l-asparaginase in 1 dog, and CHOP, l-asparaginase, lomustine, mechlorethamine, vincristine, procarbazine, and prednisone in 1 dog.

Immunohistochemical analysis—Positive FR expression was confirmed in the positive-control canine renal proximal tubular epithelial cells (Figure 1). However, definitive FR expression was not detected in tumor cells in any of the archival or prospectively collected lymphoma biopsy specimens. In some biopsy specimens, scattered individual cells were immunoreactive to PU17, and these were interpreted to possibly represent tumor-associated macrophages expressing FRβ. However, the subcellular localization of immunoreactivity was cytoplasmic, rather than at the cell membrane. Therefore, whether these cells truly expressed FRs or whether the immunoreactivity to PU17 was nonspecific was not clear.

Figure 1—
Figure 1—

Representative photomicrographs indicating results of immunohistochemical staining with PU17 polyclonal antibody to detect FRs in dogs with multicentric lymphoma. A—Section of normal canine kidney tissue used as a positive control specimen. There is intense immunoreactivity to PU17 on the apical surface of proximal tubular epithelial cells, which corresponds to the expected location of FRa. B—Section of a lymph node biopsy specimen stained with PU17. There are scattered cells with diffuse cytoplasmic immunoreactivity to PU17; these may represent tumor-associated macrophages expressing FRb. C—Section of a negative control sample stained with nonimmune serum. Bar = 100 μm.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.187

Nuclear scintigraphy—Nuclear scintigraphy revealed tumoral uptake of 99mTc-EC20 in 6 of 10 dogs (Figure 2); all 6 of these dogs had DLBCL. The 4 remaining dogs (2 dogs with DLBCL, 1 dog with marginal zone lymphoma, and 1 dog with PTCL-NOS) had no tumoral uptake of 99mTc-EC20. Five of the 6 dogs with FR-expressing tumors subsequently received EC0905, and the remaining dog received CHOP per the owner's decision. Nuclear scintigraphy was repeated at the time of disease relapse or progression in 3 of these 6 dogs with FR-expressing tumors; 2 of these dogs had received EC0905, and 1 had received treatment with CHOP. Persistent 99mTc-EC20 uptake was evident in the 2 dogs treated with EC0905, whereas uptake was absent at the time of disease relapse in the dog treated with CHOP (Figure 3).

Figure 2—
Figure 2—

Right lateral scintigraphic images of 2 dogs with DLBCL that had (A) and did not have (B) uptake of 99mTc-EC20 during nuclear scintigraphy. In panel A, notice the uptake of 99mTc-EC20 in the mandibular (asterisk), medial retropharyngeal (dagger), and superficial cervical (double dagger) lymph nodes.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.187

Figure 3—
Figure 3—

Right lateral scintigraphic images of a dog with stage 5 (according to World Health Organization criteria) DLBCL. The dog was treated with CHOP and had complete remission for 3 months before cancer relapse occurred. A—There is nonspecific uptake of 99mTc-EC20 by the liver (asterisk) and kidneys (dagger), with accumulation of excreted radioisotope in the urinary bladder (double dagger). B—Shielding of the abdominal viscera (large black circle) allows visualization of 99mTc-EC20 uptake in the mandibular (asterisk) and superficial cervical (dagger) lymph nodes, lungs and tracheobronchial lymph nodes (double dagger), and subcutaneous fat along the dorsum (number sign). C—Repeated nuclear scintigraphy at the time of cancer relapse reveals a lack of tumoral uptake of 99mTc-EC20.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.187

FR-targeted chemotherapy—Five dogs with FR-expressing tumors (as determined by results of nuclear scintigraphy) were treated with EC0905. All 5 dogs had DLBCL; 2 of these dogs had not received previous treatment, whereas 3 dogs had been treated previously (1 dog had received prednisone, 1 dog had received prednisone and l-asparaginase, and 1 dog had received CHOP, l-asparaginase, lomustine, mechlorethamine, vincristine, procarbazine, and prednisone). An objective response to EC0905 was observed in 1 of these 5 dogs, and this dog had complete remission, which was first evident 2 weeks after initiation of chemotherapy. The other 4 dogs were considered nonresponders (stable disease in 2 dogs and progressive disease in 2 dogs). The duration of stable disease in these 2 dogs was 2 weeks and 3 weeks, respectively.

The dog with complete remission was initially receiving prednisone at the time of EC0905 administration, and the prednisone dose was gradually tapered until it was completely discontinued 3 weeks after the initiation of EC0905. This dog received the scheduled dose of EC0905 for 6 weeks, and then EC0905 was discontinued as planned. Lymphoma relapsed 3 weeks after administration of the sixth dose of EC0905. At the time of cancer relapse, nuclear scintigraphy revealed persistent uptake of 99mTc-EC20, and a second round of treatment with EC0905 was planned. Hemoabdomen was also detected at the time of cancer relapse, presumably as a result of splenic or hepatic hemorrhage, although the owner would not consent to exploratory surgery for the dog to allow us to attempt to identify the source of the hemorrhage. The dog was hospitalized, and the hemoabdomen was treated conservatively. Twenty-four hours later, the dog's condition appeared stable, and it received a seventh dose of EC0905. The dog was discharged the same day that the seventh dose of EC0905 was administered, and the owner was instructed to bring the dog back to our facility in 1 week for reevaluation. However, the dog died at home 3 days after that EC0905 treatment. The cause of death was not determined because a necropsy was not performed.

In general, EC0905 treatment was tolerated well, and treatment-related toxicoses were manageable. One dog developed grade 3 diarrhea and grade 2 anorexia following the second dose of EC0905. These toxic effects coincided with cancer progression, and EC0905 was subsequently discontinued. One dog had grade 1 anorexia and grade 2 weakness following the first dose of EC0905 (this dog was concurrently receiving prednisone at the start of EC0905 treatment). That dog's second dose of EC0905 was reduced by 10%, and the prednisone dose was gradually tapered until it was discontinued at 2 weeks. No adverse effects were apparent after the second dose of EC0905, and the dog's overall condition improved markedly following that treatment. With the presumption that the dog's anorexia and weakness were not related to EC0905 administration, the dog received the third treatment of EC0905 at a dose of 0.25 mg/kg. No adverse effects were observed after the third dose, and 4 subsequent doses at 0.25 mg/kg were administered to this dog with no evidence of treatment-related adverse effects. Therefore, we attributed the weakness in this dog to glucocorticoid myopathy and the anorexia to disease, although EC0905-related toxicoses could not be definitively ruled out.

Discussion

The most important finding for the present study was that nuclear scintigraphy identified uptake of 99mTc-EC20 by the lymphoma in 6 of 10 dogs. All 6 tumors with 99mTc-EC20 uptake were DLBCL, which raised the possibility that folate uptake is more common in certain histopathologic subtypes of lymphoma. Further evaluations of dogs with other histopathologic subtypes of lymphoma would be necessary to confirm this.

A noteworthy finding of the present study was the discordant results between nuclear scintigraphy and immunohistochemical analysis. The FRs were not detected immunohistochemically in any of the tumor tissues examined, including those obtained from dogs in which nuclear scintigraphy revealed tumoral uptake of 99mTc-EC20. This was unexpected because the same immunohistochemical protocol used in this study has been used previously to detect FRs in dogs with TCC.18 Several factors could theoretically have accounted for these discordant findings. The first possibility is that a subset of lymphoma specimens had false-negative immunohistochemical results. This could occur if mutant or variant FR genes are expressed in lymphomas and the corresponding proteins are not recognized by PU17. It is also possible that lymphomas of dogs express FR isoforms that are not recognized by PU17. For example, in addition to FRα and FRβ, 2 other FR isoforms have been characterized (FRγ and FRδ). However, the γ isoform is a secretory protein,11,14 rather than a membrane-bound protein; thus, expression of FRγ should not correlate with 99mTc-EC20 uptake in discrete tumor masses, as was detected in 6 of the dogs in the study reported here. Currently, the FRδ isoform has only been identified in regulatory T lymphocytes.22,23 If FRδ were present in lymphomas of dogs and capable of binding folate or folate-conjugated pharmaceuticals, the uptake of 99mTc-EC20 observed in some of the dogs in the present study could be explained. However, there is no evidence that FRδ is expressed in any cancers of mammals at this time; therefore, we think it unlikely that it contributed to the 99mTc-EC20 uptake observed in the dogs of this study. The antibody used for immunohistochemical detection of FRs in the present study (PU17) recognizes canine FRα, as was evident for canine kidney tissue that was used as a positive control sample. The immunoreactivity to PU17 previously detected in TCCs of dogs also likely represents expression of FRα by TCCs.18 However, the ability of PU17 to recognize canine FRβ is not clearly known. In the experience of one of the authors (CPL), PU17 does not recognize human FRβ; thus, it is possible that the antibody also does not recognize canine FRβ. Therefore, it is not possible to use immunohistochemical analysis to rule out the expression of FRβ in lymphomas of dogs at this time.

Technical problems for the detection of FRs with immunodiagnostic tests are not unique to tumors of dogs. Tumor heterogeneity, antibody nonspecificity, and poor reproducibility of immunostaining have also limited the use of immunohistochemical analysis for detecting FRs in tumors of humans.17 For these reasons, functional imaging techniques, such as nuclear scintigraphy, are the preferred method for identifying human cancer patients with FR-expressing tumors for therapeutic trials with FR-targeted drugs. The FR-targeted imaging agents (eg, 99mTc-EC20) overcome the limitations of immunodiagnostic tests because they are readily internalized by cells expressing functional FRα or FRβ. Given the substantial uptake of 99mTc-EC20 observed in tumors of 6 of 10 dogs in the present study, the lack of immunoreactivity to PU17 in all tumor samples, and the general predominance of FRβ expression relative to FRα expression in hematopoietic tumors of humans,11,14 it appears plausible that lymphomas of dogs do not express FRα and that a subset of lymphomas of dogs may express functional FRβ. However, further studies are needed for confirmation.

A second possibility regarding the discordant results for the immunohistochemical analysis and nuclear scintigraphy could have been false-positive results for the nuclear scintigraphy. For example, large numbers of tumor-associated macrophages expressing FRβ could have accounted for the folate uptake detected in some tumors. However, this was considered unlikely because the numbers of tumor-associated macrophages identified in tumor biopsy specimens were much lower than needed to result in the 99mTc-EC20 uptake observed. Uptake through another transport mechanism, such as the RFC system, also was considered unlikely. The RFC system is capable of binding only 5-methyltetrahydrofolate, and it does not bind folic acid, which is the FR-targeting moiety of 99mTc-EC20, EC0905, and other FR-targeted drugs.11,15 In contrast, FRs bind folic acid with extremely high affinity, so tumoral uptake of 99mTc-EC20 observed in the dogs of the present study was likely to be an FR-mediated phenomenon. Nonspecific uptake of 99mTc-EC20 by the liver has been reported for both dogs and humans10,18 and was evident in all dogs included in the present study (Figure 3). However, to our knowledge, nonspecific uptake of folate-conjugated radiopharmaceuticals has not been detected in normal or malignant lymphoid tissues of dogs or humans.

A more compelling line of evidence against the possibility of nonspecific uptake of 99mTc-EC20 was the complete remission after EC0905 treatment in one of the dogs. Chemotherapeutics such as EC0905 and other folate-conjugated drugs must use FRs to traverse cell membranes to exert their cytotoxic effects. The chemotherapeutic EC0905 is a prodrug that consists of a hydrophilic spacer molecule and a self-immolative linker moiety that joins folate to the cytotoxic vinca alkaloid.12,18 Because of its hydrophilic spacer moiety, EC0905 cannot passively diffuse across cell membranes but must instead enter cells by FR-mediated endocytosis. After endocytosis of the FR-EC0905 complex, the linker moiety of the EC0905 molecule is degraded in the low pH environment within the endosome, and the cytotoxic vinca alkaloid is liberated to kill the cell.10 In the highly unlikely event that EC0905 passively traverses cell membranes in a non–FR-dependent manner, the linker moiety would not be degraded. Without degradation of the linker moiety, the vinca alkaloid would remain bound to the EC0905 prodrug and be unable to exert cytotoxic effects. Therefore, the complete remission observed in response to EC0905 treatment in 1 of 5 treated dogs indicated that this dog's tumor must have expressed FRs.

Similar to results for dogs with TCC,18 EC0905 treatment was generally tolerated well by the 5 treated dogs of the present study. Only 1 of the dogs developed a grade 3 toxicosis (diarrhea), which also coincided with cancer progression, so a contribution of lymphoma to the apparent chemotherapeutic toxicosis cannot be ruled out. Although the objective response rate (1/5) was modest, this rate is the same as that reported in another study24 (3/15) for dogs with lymphoma that were treated with unconjugated vinblastine. Therefore, it is possible an improved response rate might have been achieved with a folate conjugate that incorporated a drug (eg, doxorubicin) with greater single-agent activity against lymphomas.

Nuclear scintigraphy was performed in 3 of the 6 dogs treated with EC0905 at the time that the cancer relapsed or progressed. Persistent uptake of 99mTc-EC20 was observed in 2 of these 3 tumors. These results are consistent with those observed in dogs with TCC, in which persistent FR expression was detected in tumor tissue obtained at necropsy from EC0905-treated dogs that had cancer progression following treatment.18 Collectively, these results suggest that resistance to FR-targeted chemotherapy may not be mediated by decreased FR expression and that patients with cancer progression after administration of an FR-targeted drug may have cancer remission following treatment with an alternative FR-targeted agent. Curiously, persistent uptake of 99mTc-EC20 was not observed at the time of cancer relapse in 1 dog in the present study (Figure 3). Because of owner preference, this dog had received CHOP rather than EC0905, and it is possible that CHOP treatment may have affected FR expression in this dog's tumor or that clonal selection during disease progression had favored the outgrowth of a tumor cell population that did not express FRs. Additional studies are needed to determine whether FRs may be downregulated in relapsed or resistant lymphomas, compared with expression for untreated lymphomas.

Analysis of the results reported here provided evidence for folate uptake in DLBCLs, which implied overexpression of FRs in this type of cancer. However, methods for the in vitro detection of FRs in lymphomas of dogs will require additional studies to confirm FR expression. The sensitivity of in vitro detection methods, such as immunohistochemical analysis or flow cytometric analysis, could be improved by the generation of monoclonal antibodies with greater affinity for canine FRα and FRβ, and this should be a focus of future research. It appeared that FR-targeted chemotherapy was tolerated well by dogs with multicentric lymphoma, and such treatment may induce remission in some dogs with FR-expressing tumors. Future studies should address the potential for improved antitumor activity with more potent folate-drug conjugates and explore opportunities to safely integrate FR-targeted treatments into standard chemotherapy protocols to improve overall efficacy against multicentric lymphomas without increasing therapeutic toxic effects.

ABBREVIATIONS

99mTc

Technetium Tc 99m

CHOP

Cyclophosphamide, doxorubicin, vincristine, and prednisone

DLBCL

Diffuse large B-cell lymphoma

FR

Folate receptor

PTCL-NOS

Peripheral T-cell lymphoma, not otherwise specified

RFC

Reduced folate carrier

TCC

Transitional cell carcinoma

a.

Raskin RE, Fox LE. Clinical relevance of the World Health Organization classification of lymphoid neoplasms in dogs (abstr). Vet Clin Pathol 2003;32:151.

b.

Target retrieval solution, Dako Corp, Carpinteria, Calif.

c.

Background Sniper, Biocare Medical Inc, Walnut Creek, Calif.

d.

Purdue University, West Lafayette, Ind.

e.

Universal negative control serum, Biocare Medical Inc, Walnut Creek, Calif.

f.

MACH4 Universal HRP-polymer, Biocare Medical Inc, Walnut Creek, Calif.

g.

Vector Laboratories Inc, Burlingame, Calif.

h.

Richard-Allan Scientific, Kalamazoo, Mich.

i.

Etarfolatide, Endocyte Inc, West Lafayette, Ind.

j.

EC20, Endocyte Inc, West Lafayette, Ind.

k.

MiE equine scanner H.R., Scintron VI, Elk Grove Village, Ill.

l.

EC0905, Endocyte Inc, West Lafayette, Ind.

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Contributor Notes

Dr. Naughton's present address is Indiana Veterinary Imaging, 637 Kent Ave, West Lafayette, IN 47906.

Supported in part by the American Kennel Club Canine Health Foundation (Acorn grant No. 01545-A).

Presented in abstract form at the 35th Annual Conference of the Veterinary Cancer Society, Las Vegas, October 2012.

Address correspondence to Dr. Childress (mochildr@purdue.edu).