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Clinical Cancer Research Vol. 12, 7456-7464, December 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Cooperative Activity of Cytotoxic Chemotherapy with Antiangiogenic Thrombospondin-I Peptides, ABT-526 in Pet Dogs with Relapsed Lymphoma

Anthony Rusk1, Elizabeth Cozzi2, Marty Stebbins1, David Vail3, Joanne Graham4, Victor Valli5, Jack Henkin2, Rick Sharpee2 and Chand Khanna1

Authors' Affiliations: 1 Animal Clinical Investigation, LLC, Bethesda, Maryland; 2 Abbott Animal Health, Abbott Park, Illinois; 3 University of Wisconsin-Madison, Madison, Wisconsin; 4 Arboretum View Animal Hospital, Downers Grove, Illinois; and 5 University of Illinois-Champaign Urbana, Urbana, Illinois

Requests for reprints: Anthony Rusk, Animal Clinical Investigation, LLC, at Friendship Hospital for Animals, 4105 Brandywine St., NW, Washington, D.C. 20016. Phone: 410-419-0804; Fax: 202-363-7126; E-maill: trusk{at}animalci.com.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Thrombospondin-I (TSP-I) is a natural antiangiogenic protein that enhances apoptosis of activated endothelial cells. A modified nonapeptide from TSP-I, ABT-526, has been found to be active in mouse cancer models and in dogs with naturally occurring cancers. To further assist in the development of ABT-526, we report herein on its evaluation in combination with cytotoxic chemotherapy in pet dogs with relapsed non-Hodgkin's lymphoma (NHL).

Experimental Design: Ninety-four pet dogs with naturally occurring first-relapse NHL were entered into a prospective randomized placebo controlled double-blinded trial of ABT-526 plus CeeNu (Bristol-Myers Squibb, New York, NY) versus CeeNu alone. Endpoints included response rate, duration of response, time to progression, and incidence of toxicoses.

Results: No significant ABT-526-specific toxicities were seen. CeeNu-associated toxicities, including neutropenia, thrombocytopenia, gastroenteritis, and elevated alanine transaminase, were similar. No significant difference in objective response rate was seen (ABT-526 + CeeNu versus placebo + CeeNu, 23/49 versus 23/37; P > 0.25). Cooperative activity between ABT-526 and CeeNu chemotherapy was evident based on a significant increase in the median response duration of dogs receiving ABT-526 plus CeeNu compared with placebo plus CeeNu (35 versus 15 days; P < 0.05). The time to progression for responding cases was also significantly greater in dogs receiving ABT-526 plus CeeNu compared with placebo plus CeeNu (41 versus 21 days; P < 0.05).

Conclusions: Results of this preclinical trial suggest that the activity of ABT-526 is sustained when combined with cytotoxic chemotherapy; furthermore, the activity seems to be associated with the maintenance of CeeNu-induced treatment responses. Further studies of TSP-I peptide antiangiogenic therapy in pet dogs and humans with NHL are warranted.

The prevention of new blood vessel formation or targeting existing tumor-associated blood vessels is a novel approach for the treatment of cancer (1–5). Natural protein inhibitors of angiogenesis include thrombospondin-I, metallospondins, and proteolytic degradation products such as angiostatin, endostatin, kringle 5, and vasostatin (6). Pleiotropic inhibitors such as thrombospondin-I are capable of blocking several aspects of endothelial cell activation initiated by most of the known growth factors. Although individual endothelial cell activators operate via specific cell receptors, all seem to have similar cell signaling pathways. Recently, a common mechanism has been suggested for the natural inhibitors (7) in which each inhibitor, using its unique receptor, leads to apoptosis of the activated endothelial cell. Because the inhibitors have no effect on quiescent endothelial cells, activation is required to prime the cell for induction of an apoptotic program by these inhibitors.

Thrombospondin-I (TSP-I) was the first discovered natural angiogenesis inhibitor (8). TSP-I and the closely related TSP-II (9) have the ability to act broadly against a wide variety of proangiogenic growth factors; however, their large molecular size and multifunctional nature have precluded their use as therapeutic agents. Modified peptide segments of the antiangiogenic domain of TSP-I containing D-amino acids have been shown to mimic the antiangiogenic action of TSP-I (10). Recently, two further modified nonapeptides in this series, ABT-526 and ABT-510 (DI-TSP and DI-TSPa, respectively), have shown similar antiangiogenic activity. In vitro studies have shown that 0.5 to 10 nmol/L concentrations of these peptides block the migration of human microvascular endothelial cells stimulated by several growth factors, abrogate endothelial cell proliferation and tube assembly in fibrin gels stimulated by vascular endothelial growth factor, and enhance apoptosis of activated endothelial cells (9, 11). In vivo, both ABT-526 and ABT-510 slow tumor growth in syngeneic and xenograft mouse models, and, like TSP-I (7), these peptides increase the apoptotic index of endothelial cells at the periphery of tumors in orthotopic mouse models (11). To determine if TSP-I peptides were active against spontaneously arising tumors, pet dogs with measurable malignant cancers were treated with single agent ABT-526.6 TSP-I peptides were active against canine endothelial cells in vitro and were associated with pharmacokinetic profiles similar to those seen in mice and humans. The mean elimination half-life for ABT-510 and ABT-526 was 0.7 to 0.8 h (range, 0.5-1 h). No adverse reactions or toxicity were noted in any dogs treated in this open-label single-agent study of 54 dogs. Objective regressions of measurable tumors were seen in 10 of 54 (18%) dogs receiving ABT-526 therapy for a minimum of 30 days. ABT-526-associated responses were seen in several cancer types including dogs with chemotherapy-resistant non-Hodgkin's lymphoma (NHL). Results from this open-label trial defined NHL as one of the more responsive histologies. These preclinical data contributed to the entry of TSP-I peptides into phase I human clinical trials (12).

Many questions relating to the development path of TSP-I peptides as they move towards phase II clinical trials remain. These include optimal biological dose, schedule and regimen, definition of responsive histologies, activity of TSP-I agents in combination with conventional therapies, and predictive biological markers for activity. It is not likely that all questions can be answered within human phase I trials or through conventional mouse models of cancer. To contribute to the human clinical development of TSP-I peptides and to define the value of TSP-I peptides as a treatment for pet dogs, a randomized prospective trial of ABT-526 in combination with single-agent lomustine (CeeNu Bristol Myers Squibb) chemotherapy for dogs with relapsed NHL was initiated. The results of the trial reported herein support cooperative activity, without additional toxicity, of ABT-526 with CeeNu chemotherapy in the treatment of relapsed NHL. ABT-526 did not increase the number of cases responding compared with placebo + CeeNu; however, the duration of response was modestly enhanced by ABT-526 exposure. Information from this trial will be informative as TSP-I peptides advance in clinical development.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study design. The study was designed as a randomized, placebo-blinded trial for pet dogs with first-relapse NHL. Pet dogs were evaluated at one of 15 specialized veterinary oncology centers in the United States (see Acknowledgments). Eligibility criteria included a histologic or cytologic diagnosis of NHL (later confirmed by histology); a first complete remission duration of at least 4-month duration; no prior treatment with CeeNu; discontinuation of induction chemotherapy for at least 21 days before study initiation (glucocorticoid or nonsteroidal anti-inflammatory therapy was permitted only if patients had received this therapy longer than 30 days before study enrollment); presence of first relapse, defined by palpation or radiographic assessment of measurable lymph nodes; performance status of 0, 1, or 2 (based on a modified Eastern Cooperative Oncology Group grading scheme; ref. 13); hematologic status grade 0 or 1 (Table 1 ); serum creatinine concentration <4.0 mg/dL; body weight >10 kg (22 pounds); and written informed consent provided by the owner(s) of each dog. A complete history, physical examination, and two-dimensional measurements of up to three representative lymph nodes, complete blood count, platelet count, serum biochemical analyses, urinalysis, thoracic and abdominal radiography, and lymph node biopsy were done before treatment. Clinical stage was determined on the basis of established criteria (13).


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  		Table 1. Hematologic grade

 
Randomization. Dogs were initially randomized into three treatment groups: A, B, and C. Group A was assigned to receive CeeNu (70 mg/m2) plus ABT-526 at 0.8 mg/kg/d. Group B received CeeNu (70 mg/m2) plus ABT-526 at 0.4 mg/kg/d. Group C received CeeNu (70 mg/m2) plus placebo. A balanced randomization scheme using a random permuted block method was implemented. Within each group, dogs were stratified by type of induction chemotherapy protocol [cyclophosphamide-Adriamycin-vincristine-prednisone (CHOP)–like; University of Wisconsin (UW)-Madison–like or Panel Comparability (PACO)-prednisone, Adriamycin, cyclophosphamide, Oncovin, prednisone, and leukaran (PAL)–like; or other; Fig. 1 ]. Treatment institution was not used as a randomization strata. After slow initial accrual to this study, accrual to group B was discontinued. Accordingly, only five cases were entered to randomization group B. All subsequent cases were randomized to groups A and C (1:1), following a brief slanted randomization of A/C 3:1. Treatment group assignments were done by a trial monitor (JT) not directly involved in patient care. Clinicians and owners were blinded to treatment group assignment.


Figure 1
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  		Fig. 1. Randomization and stratification schema used in for prospective preclinical trial of ABT-526 antiangiogenic therapy plus CeeNU versus CeeNU alone. Strata were defined based on the type of induction chemotherapy used in the management of dogs at initial diagnosis. Dogs were required to have a first remission duration of at least 4 months using one of these induction protocols to be eligible for this relapsed NHL study.

 
Agents. ABT-526 was supplied by Abbott Laboratories (Abbott Park, IL) in vials containing 0.5 or 1.0 mL of 12.5 or 25 mg/mL ABT-526 in dextrose. Animals assigned to group A (0.8 mg/kg ABT-526) received material labeled as "treatment A" (1.0 mL/vial). Animals assigned to group B (0.4 mg/kg ABT-526) received material labeled as "treatment B" (0.5 mL/vial). Doses were rounded up to the nearest 0.1 mL. Placebo was supplied by Abbott in vials labeled "treatment C" containing 2 mL of 0.9% sodium chloride (Abbott). CeeNu was supplied by the investigators.

Endpoints. Complete response was defined as disappearance of all clinical evidence of cancer (both target and nontarget lesions) and of any signs related to the cancer. Partial response was defined as a ≥50% decrease in the sum of the products of measurements for target lesions; minor response was defined as a 25% to 50% decrease in the sum of the products of measurements for representative lesions. Stable disease was defined as any reduction in target lesions less than that defined for minor response or any progression not defined as progressive disease. Progressive disease was defined as an unequivocal increase of at least 50% in the size of any measurable lesion or appearance of new lesions. Relapse was defined as appearance of new lesions or reappearance of old lesions in dogs that had had a complete remission; in dogs that had had only a minimal or partial response, relapse was defined as at least a 50% increase in the sum of the products of measurements of target lesions, compared with measurements obtained at the time of maximum response. Response was defined as the time from at least a minor response to relapse. Time to progression was defined as the time from study initiation to relapse or progressive disease. For this study, measurement of no more than three representative lesions was defined as target lesions, and followed with caliper measurements.

Study outcomes compared between treatment groups A and C included objective response rate (i.e., the proportion of dogs in a study group achieving a partial response or complete response), biological response rate (i.e., the proportion of dogs in a study group achieving a minor response, partial response or complete response), time to progression (i.e., number of days from the onset of treatment to the first day disease progression or relapse), duration of biological response (i.e., number of days from the onset of biological response to the first day of relapse or disease progression), duration of objective response (i.e., number of days from the onset of biological response to the first day of relapse or disease progression), survival time (i.e., number of days from the onset of treatment to death, regardless of whether death was related or unrelated to NHL), and number of episodes of dose-limiting toxicity during treatment. Toxicity end points included the number of episodes of dose-limiting neutropenia and thrombocytopenia during treatment, number of dose-limiting episodes of gastrointestinal toxicity during treatment, and number of dogs in which death was associated with treatment. At the time of progressive disease or relapse, cases could receive any treatment determined appropriate by the primary care veterinarian. Continued follow-up was requested on all cases including type of salvage chemotherapy, remission rate, duration of subsequent remission, and survival. Necropsy examination was recommended in all cases.

Treatment protocol. All cases received CeeNu every 21 days for a maximum of five treatments and ABT-526 or placebo as a s.c. injection divided twice daily. Cases with stable disease, minor response, partial response, or complete response after the fifth dose of CeeNu were allowed to continue with s.c. injections of study drug. On days 0, 7, 14, and 21, and every 21 days thereafter, target lesions were measured. At each visit, owners were queried on quality of life using a quality-of-life survey (Supplementary Information I) and for the presence of toxicoses (e.g., vomiting, diarrhea, or anorexia). Complete blood count was evaluated before and 7 and 21 days following each treatment with CeeNu. Alanine transaminase was measured before each treatment with CeeNu. If drug-induced neutropenia (i.e., grade 3 or 4 granulocyte status; Table 2 ) was detected at day 7, future treatments with CeeNu were given at a 25% dose reduction for the remainder of the study. If drug-induced neutropenia (i.e., grade 2, 3, or 4 granulocyte status; Table 2) was detected immediately before a scheduled CeeNu treatment, then CeeNu treatment was delayed for 7 days. Subsequent treatments with CeeNu were reinitiated at a 25% dose reduction after a return to granulocyte status of grade 0 or 1. If platelet counts were <75,000/µL following CeeNu therapy, then study treatments were discontinued. If the baseline platelet count, collected at the time of study entry, was <75,000/µL, cases were permitted to continue on-study if the platelet count did not decrease below the baseline count. If severe drug-induced gastrointestinal toxicity developed, CeeNu was withheld until 3 days after signs resolved and subsequent dosages of CeeNu were reduced by 25%. If serum alanine transaminase levels increased to >2-fold over laboratory reference range or the patient baseline, for dogs with elevated alanine transaminase, at the start of therapy after CeeNu administration, then study treatments were discontinued. All deviations from study eligibility criteria, dosing instructions, and follow-up were recorded.


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  		Table 2. Granulocyte grade

 
Pathology. All diagnoses were confirmed and graded by a single pathologist (T.V.) The tissues were processed routinely for paraffin embedding and were cut in one step at 5 µm for all three stains, H&E, CD79{alpha}, and CD3. Cases with CD3-negative immunophenotype were preferentially recruited; however, CD3-positive status was not an exclusion criteria. Antigen retrieval was carried out by heating the mounted deparaffinized tissues in EDTA at pH 6 for 10 min. Diagnostic antibodies were from DAKO and the 3,3'-diaminobenzidine reaction system was used. All tissues were well fixed and processed with good cell detail but, in some of cases, the diagnosis was made on small tissue fragments. The tissue diagnosis was based on the revised WHO classification system that identifies subtypes of lymphoma as disease entities rather than solely on cell type. To accommodate the statistical evaluation, the list of diagnoses was divided into a set of four grades with grade 1 as the most indolent types and grade 4 as the most aggressive types. The mitotic rate of each case was determined by counting the number of cells at any stage of mitosis per field at x400 magnification. Notations were also made of the number of nuclei undergoing apoptosis and the number of tingible body macrophages in the same areas as the mitoses were noted. Generally, two to three areas from each case were counted except where the tissues were too small or were of mixed benign and neoplastic types. Cell count was not possible in five cases. For small cell lymphomas, the level of mitoses is a very important variable in recognizing the lymphoblastic lymphomas that otherwise might be considered intermediate type of relatively indolent small cell neoplasms. A further additional notation was made for large B-cell lymphoma depending on the nuclear size and the nucleolar arrangement. Neoplasms with a predominant pattern of a single prominent central nucleolus were called immunoblastic type. Those with multiple nucleoli impinging on the nuclear membrane were called centroblastic, describing benign nuclei of the germinal centers and malignant nuclei of follicular lymphoma. If there were mixtures of immunoblastic and centroblastic nuclear types, these were noted as immunoblastic/centroblastic if the immunoblastic type predominated and centroblastic/immunoblastic if the centroblastic type was more numerous.

Grading system for canine lymphomas was based on a modification of National Cancer Institute working formulation:

Grade 1
All follicular lymphomas grades 1/2/3
Marginal zone lymphoma
Mantle cell lymphoma
Small cell lymphocytic lymphoma

Grade 2
Intermediate type B-cell lymphoma
Centrocytic cell lymphoma
T-cell–rich large B-cell lymphoma

Grade 3
Centroblastic cell lymphoma
Immunoblastic cell lymphoma
Plasmablastic B-cell lymphoma

Grade 4
Lymphoblastic B-cell lymphoma
Lymphoblastic T-cell lymphoma including convoluted types

Study analysis included dogs randomized to group A and group C only (Fig. 2 ); group B patients were not included in this analysis. The intent-to-treat population was defined as all dogs that were randomized and initiated therapy for a minimum of 7 days. The treatment-received population included only those dogs that were able to remain progression-free for 21 days or longer.


Figure 2
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  		Fig. 2. Randomization of cases to prospective preclinical trial of ABT-526 antiangiogenic therapy plus CeeNU versus CeeNU alone. Study analysis included two populations that included dogs randomized to group A and group C only. Group B patients were not included in this analysis or this report. The intent-to-treat population included all dogs that were randomized and initiated therapy. The treatment-received population included only those dogs that were able to remain progression-free for 21 d or longer. Dogs that withdrew from treatment before 7 d of study were not included in the analysis of either study population.

 
Statistics. Prestudy estimates of sample size suggested that 31 dogs with non–T-cell NHL were sufficient to measure a 50% increase (25 days) in the time to progression between treatment groups, assuming a mean time to progression of 50 days and a 35-day standard deviation, with a P value of 0.05 and power of 0.80. Estimates of mean time to progression and standard deviation were estimated based on recent reports of monotherapy chemotherapy for relapsed canine NHL (14, 15). A target accrual size of 35 cases of non–T-cell NHL for each treatment group was estimated to be sufficient for successful randomization and follow-up of the target number of non–T-cell NHL cases. Forty-six cases of NHL (B- and T-cell) per treatment group were estimated to be sufficient to provide the target accrual of 35 non–T-cell NHL cases.

Descriptive statistics included the use of nonparametric (Kruskal-Wallis) and alternate (Mann-Whitney) t tests and Fisher's exact t test. P < 0.05 was considered significant. Time to disease progression, duration of remission, and survival time were compared between groups using log-rank survival analysis. Univariate analysis examined differences with regard to stratification variables (induction therapy protocol) and other potential confounders (clinical stage, clinical substage, histologic grade, and immunophenotype) between treatment groups and between biological responders and nonresponders. Only histologically confirmed cases of malignant NHL were considered in the final evaluation.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Population description. A total of 95 cases entered the study. A single case was randomized for study but withdrew before receiving any therapy and was not included in the analysis. Forty-nine dogs were randomized to treatment A (ABT-526 + CeeNu), 5 dogs to treatment B, and 39 to treatment C (placebo + CeeNu). The distribution of cases following randomization is summarized in Fig. 2. The unequal assignment to treatments A and C was the result of "chance" alone and not directed by stratification. Table 3 provides a summary of the characteristics of dogs entering this trial using known or potential covariates for outcome in dogs with NHL.


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  		Table 3. Characterization of dogs randomized to ABT-526 + CeeNu versus placebo + CeeNu for known and potential covariates of outcome in canine lymphoma

 
Pathology. A total of 88 lymph node samples were available for histologic examination (Table 3). The lymphoma subtypes included in grade 1 included marginal zone lymphoma (13 cases), mantle cell lymphoma (1 case), follicular lymphoma (7 cases), small cell lymphocytic lymphoma (3 cases), and small cell intermediate (1 case). There was also 1 case of small cell lymphocytic lymphoma of T-cell type. Collectively, there were 24 (27%) indolent lymphomas of B-cell differentiation. In contrast, the lymphoma subtypes included in grade 4 included lymphoblastic of B-cell type (7 cases) and lymphoblastic of T-cell type (4 cases). Collectively, there were 11 (13%) high-grade lymphomas of B-cell and T-cell differentiation.

An established predictor of outcome in canine NHL is the dose intensity of cytotoxic chemotherapy. Data comparing dose-intensity and the related variables of dose limiting toxicities associated with therapy are listed in Table 3. Treatment with ABT-526 was found to be safe and well tolerated when combined with CeeNu chemotherapy in dogs with first-relapse NHL. No difference in dose-limiting neutropenia, thrombocytopenia, and gastrointestinal or hepatic toxicity was seen in dogs treated with CeeNu + placebo versus CeeNu + ABT-526. Quality of life assessment, defined by a questionnaire completed by pet owners at each pet visit, did not reveal differences in quality of life in dogs receiving CeeNu + placebo versus CeeNu + ABT-526 (data not shown). Serial quality-of-life surveys were available for 24 patients (71 reports) receiving CeeNu + placebo and for 37 patients (117 reports) receiving CeeNu + ABT-526. Although not statistically significant, the frequency of owners reporting "rare discomfort" following s.c. injection was greater in dogs receiving ABT-526 (16.1%) versus placebo (4.3%). This is likely the result of dextrose in treatment A (ABT-526 formulation). A potential adverse event associated with ABT-526 exposure was seen in a single dog that experienced acute bilateral conjunctivitis and anterior uveitis. These ocular changes occurred contemporaneously with lymphoma progression and therefore cannot be definitively associated with ABT-526 exposure.

Of the examined covariates (listed in Table 3), age of dogs was the only one found by univariate analysis to have a statistically significant relationship with outcome (Table 4 ). Dogs older than 9 years had a statistically poorer response rate than dogs younger than 9 years (relative risk, 1.93; P = 0.022). The lack of statistically significant associations with any other examined covariate argued against the value of multivariate modeling. These results suggest that unknown covariates of outcome were presumably balanced equally between treatment groups. This balanced randomization provided confidence that the analysis of outcome, reported below, as a function of treatment group assignment is the result of treatment assignment rather than an influence of an unknown covariate. The absence of an association between outcome and clinical substage b and T-cell immunophenotype, previously shown to be risk factors for canine NHL, is likely the result of small sample numbers in these subgroups, a result of study design, which excluded most dogs with these attributes.


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  		Table 4. Univariate analysis of intent-to-treat study population for known and potential covariates of outcome in canine lymphoma

 
Objective and biological responses and response duration. Response to therapy is a significant prognostic factor for dogs with NHL and is a conventional end point used in the assessment of cytotoxic therapies for NHL (16). Objective response, including both partial response and complete response, is a recognized determinant of the activity of a cytotoxic therapeutic agent. The term biological response has been used more recently to assess novel, potentially cytostatic agents. Biological response includes minor, partial, and complete responses. Table 4 shows statistically significant associations between complete response, objective response, and biological response and response duration for all dogs in this study, irrespective of treatment group assignment (i.e., independent of exposure to ABT-526). Table 5 includes a comparison of response rates and response duration between dogs randomized to receive ABT-526 + CeeNu versus placebo + CeeNu in the intent-to-treat dog population. No differences in the proportion of dogs achieving either objective response or biological response (response rates) were seen between dogs receiving ABT-526 + CeeNu and those receiving placebo + CeeNu. The median duration of objective response for dogs receiving ABT-526 + CeeNu was significantly longer (133%; 35 versus 15 days; P < 0.050) than for dogs receiving placebo + CeeNu. The duration of biological response for dogs receiving ABT-526 + CeeNu was also longer (93.1%), however, without statistical significance (P = 0.13).


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  		Table 5. Comparison of objective and biological response rates and response duration between dogs receiving ABT-526 + CeeNu versus placebo + CeeNu

 
Time to progression in responding population. Response to therapy may be an inappropriate end point for the evaluation of a novel noncytotoxic agent like ABT-526. The benefit of ABT-526 and other antiangiogenic agents may include disease stabilization or delays in tumor progression. Such benefits would not be captured by conventional response end points. As such, alternative end points including significant stabilization of disease and time to progression have been suggested for clinical trial evaluation (Response Evaluation Criteria in Solid Tumors criteria; ref. 17). In Table 6 , time to progression for dogs achieving a biological response is reported for dogs receiving ABT-526 + CeeNu versus placebo + CeeNu in both the intent-to-treat and treatment-received populations. For the intent-to-treat and treatment-received populations of dogs, a statistically significant (P < 0.05) and nearly significant (P = 0.057) increase, respectively, in median time to progression was seen for dogs receiving ABT-526 + CeeNu compared with CeeNu alone. The increase in time to progression for responding cases receiving ABT-526 was ~20 days in both study populations. No significant differences in time to progression were seen between ABT-526 + CeeNu and placebo + CeeNu in either the intent-to-treat or treatment-received population unless analysis of the responding population was examined (not shown).


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  		Table 6. Comparison of time to progression in biological responders in both intent-to-treat and treatment-received populations for dogs receiving ABT-526 + CeeNu versus placebo + CeeNu

 
Survival. Following progression of disease, >80% of dogs randomized to this study received conventional rescue chemotherapy. The type of follow-up chemotherapy was variable. There was no difference in overall survival seen between dogs receiving ABT-526 + CeeNu and dogs receiving ABT-526 alone (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Results of this nonclinical study in dogs with naturally occurring cancer suggest that TSP-I peptides are active anticancer agents when combined with cytotoxic chemotherapy and are not associated with additive side effects. The benefit of TSP-I peptides shown in this study is associated with extension of the response duration provided by cytotoxic chemotherapy.

The clinical development of antiangiogenic agents for cancer has been complicated and not adequately informed by conventional preclinical cancer models (i.e., transplantation mouse models; ref. 18). The biology of angiogenesis involves a complex interaction between host, tumor, and microenvironment that is difficult to model in murine cancer xenografts (reviewed previously in refs. 19–21). An alternative and informative translational modeling approach involves the study of cancer treatment agents in prospective clinical trials in companion animals (i.e., pet dogs that naturally develop cancers; ref. 20). Examples of such cancers include NHL, prostate carcinoma, lung carcinoma, head and neck carcinoma, mammary carcinoma, melanoma, soft tissue sarcoma, and osteosarcoma. The rationale, strengths, and weaknesses of translational and biology-based studies in cancer using companion animals have recently been reviewed (20). For agents that have not entered the clinic, companion dog studies may provide a combination of toxicity and efficacy data that may support initiation of phase I studies in humans (22). For agents that are currently in phase I exploration in humans, pet dog studies may be able to define optimal treatment regimens, uncover uniquely sensitive histologies, or validate biomarkers that will then be useful in phase II investigations in humans (23). It is in this setting that this nonclinical study of ABT-526 was initiated. Results of phase I studies in humans were encouraging and supported the consideration of phase II studies. Not surprisingly, data from phase I human studies alone could not answer all questions that could best direct phase II studies. Single-agent activity of ABT-526 was shown in dogs with NHL; however, no data on the activity of ABT-526 in combination with chemotherapy in NHL were available. NHL in dogs is the most common malignant cancer diagnosed and treated in veterinary practice. The histology, biology, and therapy for lymphoma are similar in dogs and humans. Where assessed, the molecular changes that characterize both canine and human NHL have also been found to be similar. Additional studies to define the molecular characteristics of canine lymphoma are under way (24, 25). If untreated, dogs with NHL are likely to experience life-threatening progression of disease within 30 to 45 days of diagnosis (26). Treatment of NHL in dogs, like in humans, most commonly consists of multiagent, CHOP-like chemotherapy (27). Most chemotherapy protocols induce remission (complete response) in >80% of cases (16) with median response durations of ~1 year. Relapsed cases of NHL in dogs may be successfully treated with combination chemotherapy; however, they are less likely to achieve remission (objective response rate, 50%) than at initial diagnosis (14, 28, 29). The development of chemotherapy resistance, in part, explains the relative difficulty associated with treating NHL in both dogs and humans. These similarities between dogs and humans have been the basis for the conduct of nonclinical trials of novel cancer treatment agents in companion dogs.

In this study of dogs with relapsed lymphoma, single agent CeeNU was used alone or in combination with antiangiogenic peptides of ABT-526. The rationale for selection of CeeNU was based on its shown efficacy in the relapse setting in dogs (29). The anticipated outcome associated with single agent CeeNU was thought to be predictable and likely to allow the timely assessment of any benefit associated with ABT-526 efficacy. Results from this trial in dogs with first-relapse NHL support the activity of ABT-526. Although not initially considered to be a histology sensitive to antiangiogenic agents, recent data continue to support that lymphomas and leukemias are dependent on angiogenesis. The antilymphoma activity shown in this study was best shown in dogs that were responsive to CeeNu chemotherapy. It is possible that responses to CeeNu slowed the rapid progression associated with NHL relapse and then allowed ABT-526 to control or slow disease progression through an antiangiogenic mechanism. It is also possible that the determinants for responsiveness to CeeNu are the same as the determinants for responsiveness to ABT-526. To determine if clinical variables collected in this study could define a CeeNu-responsive (and potentially ABT-526-responsive) profile for dogs with relapsed NHL, univariate analysis for statistical association between outcome (objective response and time to progression) and age, sex, initial chemotherapy protocol, first remission duration, stage of disease and substage, histologic grade, and immunophenotype was undertaken. In this analysis, only age of dogs was predictive of outcome. Similar age-associated influences on outcome have been reported in NHL in dogs (30). It is unlikely that age of dogs is linked to antiangiogenic responsiveness of dogs with NHL. It is interesting that histologic grade was not predictive of outcome in this population of dogs. The assessment of histologic grade as a predictor for response or outcome in dogs with NHL has previously been reported; however, this is the first prospective report that included histologic assessment at the time of relapse. It is not likely that the analysis of cellular features of NHL alone will define populations as either responsive or not responsive to conventional or novel cancer therapies like antiangiogenic TSP-I mimetic peptides. The opportunity for pre- and post-treatment lymph node biopsy from pet dogs included in clinical trials of novel cancer drugs was not realized in this study. Future translational studies that include dogs with cancer should take advantage of the opportunity for such serial biopsies. Additional information including cellular proliferation and genomic and proteomic profiles of subgroups of dogs with NHL are under way. At this time, it is not possible to predict which cases are most likely to benefit from ABT-526 therapy. Such a priori markers are essential for the successful development of human cancer drugs. Work is under way to define and validate such biomarkers in tumor-bearing dogs exposed to TSP-I peptides.

The reported objective response rate for cytotoxic chemotherapy protocols used for relapsed NHLs ranges from 40% to 90% (14, 29). A single report of 43 dogs treated with monotherapy CeeNu found objective response in 11 (25%) dogs with relapsed NHL. The median duration of response in the responding population was 86 days (29). This previous report included dogs with first-, second-, and third-relapse NHLs. Anecdotal experience with CeeNu treatment for dog with first-relapse NHLs suggested an objective response rate of ~50%. This data was used to predict outcome for dogs treated with CeeNu alone in prestudy sample size calculations. The estimated time to progression for dogs receiving CeeNu was 50 days. As noted in Tables 4 and 5, the response rates associated with CeeNu alone matched prestudy estimates; however, the duration of responses was notably shorter than expected. This is may be the result of a 22% decrease in CeeNu dose intensity used for this study compared with previous reports in the literature (70 versus 90 mg/m2). Alternative explanations may include differences in study population or the small sample sizes of previous reports in the literature. The effect of the shorter-than-expected remission duration associated with CeeNu alone is significant. The statistically significant improvement in outcome associated with ABT-526 exposure (measured by duration of objective response) when measured in days (20 days) is small. However, had the CeeNu response duration met expectations of 50 to 60 days, the percent increase in remission duration seen for dogs exposed to ABT-526 may have resulted in a clinically as well as statistically significant improvement in ABT-526-associated outcome. Based on the results of this study, future studies in lymphoma using ABT-526 in dogs with lymphoma should include a more active cytotoxic chemotherapy protocol on which a broader and more long-term benefit of ABT-526 may be seen. Alternatively or in addition, a trial focusing on a more chemotherapy-responsive population of dogs with NHL (i.e., dogs at diagnosis who are naïve to all chemotherapy) may be informative. It is important to note that the addition of a TSP-I peptide to a conventional cytotoxic chemotherapeutic agent did not augment toxicity normally associated with that agent (i.e., CeeNU).

Data generated in this dog study suggest that combination of TSP-I peptides and cytotoxic chemotherapy may be safely used with potential cooperative activities, and that NHL may be a sensitive histology for TSP-I peptide antiangiogenic therapy. It seems that the benefits of TSP-I peptides therapy are likely to be most notable when combined with treatment modalities that control rapid primary tumor progression. Future studies in dogs may be useful to determine if TSP-I peptides may be safely combined with other convential cytotoxic agents, with radiation therapy, or with novel anticancer agents.


   Acknowledgments
 
We thank the collaborative Network of veterinary oncologists and veterinary nurses, including the Animal Clinical Investigation Oncology Network San Francisco Veterinary Specialists (Dr. C. Rodriguez), University of Wisconsin-Madison, School of Veterinary Medicine (Dr. D. Vail), Pet Emergency and Specialty Center (Dr. B. Phillips), Southwest Veterinary Oncology and Arizona Veterinary Specialists (Drs. M.K. Klein and B. Hershey), Arboretum Veterinary Referral (Dr. G. Graham), Veterinary Specialty Center of Colorado (Dr. R. Elmslie), Gulf Coast Veterinary Oncology (Dr. K. Hahn), Animal Medical Center (Dr. P. Bergman), Veterinary Hospital of the University of Pennsylvania (Dr. K. Sorenmo), The Oncology Service–Friendship Hospital for Animals, Southpaws Veterinary Referral (Dr. Sheaffor), Veterinary Referral Associates—VRA (Dr. Fulton), Beltway Veterinary Referral (Drs. L. Bravo and K. Arrington), and Atlantic Veterinary Referral (Dr. J. Peterson) for outstanding care of pet animals involved in this study; Jennifer Turner and Kate Cadorette for trial management and oversight, and the pet owners and patients entered to this study.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

6 Please see companion article, A. Rusk, et al., Preclinical evaluation of antiangiogenic thrombospondin-1 peptide mimetics, ABT-526 and ABT-510, in companion dogs with naturally recurring cancers, in this issue. Back

Received 1/17/06; revised 4/23/06; accepted 9/22/06.


   References
Top
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 Materials and Methods
 Results
 Discussion
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