Influence of Asparaginase on a Combination Chemotherapy Protocol for Canine Multicentric Lymphoma

Introduction

Lymphoma is a common cancer of dogs that is typically responsive to chemotherapy.¹ Many chemotherapy protocols have been developed over the past two decades, with complete remission (CR) rates between 60% and 90%, and initial remission durations of 6 to 8 months.¹ Contemporary chemotherapy represents various additions to the cyclophosphamide, vincristine, prednisone (COP) protocol, originally described 20 years ago.² Although combination chemotherapy protocols are superior to single-agent protocols for the treatment of canine lymphoma, the contributory effect of each individual drug used within a protocol remains unknown. The efficacy of the single-agent asparaginase^a for the treatment of canine lymphoma has been reported, and asparaginase has been incorporated into various combination chemotherapy protocols for canine lymphoma.^1,3–13

Asparaginase (also known as L-asparaginase) is a high molecular-weight (133 Kd) enzyme purified from bacterial sources.¹⁴ The preparation commonly used in the clinical setting is derived from Escherichia coli. This enzyme hydrolyzes asparagine into aspartic acid and ammonia, and glutamine into glutamic acid.^14–17 The result is a rapid inhibition of protein synthesis and delayed inhibition of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis. Asparaginase is most active in the growth phase 1 (G₁) of the cell cycle.¹⁸ Its effect on lymphoid malignancies arises from the inability of lymphoblasts to synthesize asparagine from aspartic acid.^17,19 Asparaginase induces CR in up to 60% of humans with acute lymphoblastic leukemia and has been widely used for canine lymphoma.^{1,3–13,20,21} The purpose of this study was to determine if L-asparaginase increases the clinical response rate and prolongs the initial progression-free interval (PFI) when added to a modified COP chemotherapy protocol for canine lymphoma.

Materials and Methods

A computerized search of medical records was done to identify dogs with lymphoma treated between 1987 and 2003 at the Purdue University Veterinary Teaching Hospital. Dogs with multicentric lymphoma were eligible for the study only if they were evaluated with a physical examination, ophthalmic examination, complete blood count, serum biochemical profile, urinalysis, thoracic and abdominal radiography, abdominal ultrasonography, bone marrow aspirate and cytology, lymph node histopathology, and measurement of peripheral lymph nodes.

Dogs included in the study were treated with a similar induction chemotherapy protocol consisting of cyclophosphamide,^b vincristine,^c and prednisone^d [Table 1]. Some dogs were also given one or two doses of asparaginase (COPA). Whether a dog received zero, one, or two doses of asparaginase was decided randomly and solely depended upon the availability of the drug at the time of diagnosis and treatment. The induction chemotherapy protocol was repeated for 6 consecutive weeks. All dogs received identical maintenance chemotherapy [Table 2], consisting of methotrexate,^e prednisone, and cyclophosphamide. The maintenance chemotherapy protocol was given for 6 weeks followed by 1 week of the induction chemotherapy protocol. This cycle of 6 weeks of maintenance chemotherapy, followed by 1 week of induction chemotherapy, was repeated until relapse occurred.

Dogs were excluded from the study if they did not undergo all the aforementioned diagnostic procedures. Dogs with other anatomical forms of lymphoma were also excluded from the study. Dogs that exhibited a low tolerance for certain drugs within the chemotherapy protocol and required substitute chemotherapeutic agents were excluded from the study.

Complete remission was defined as the resolution of all evidence of lymphoma. Partial remission (PR) was defined as ≥50% decrease in lymph node volume. Stable disease referred to <50% decrease in lymph node volume, and progressive disease denoted ≥50% increase in lymph node volume or the development of new tumor lesions. The PFI was defined as the interval between the date of first remission and the date of first relapse.

The two chemotherapy treatment groups were compared with respect to sex, weight, age, and World Health Organization (WHO) clinical stage and substage frequency. Remission status at 2 weeks and 6 weeks of induction chemotherapy, and PFI were compared between groups. Toxicity between the protocols was not evaluated because of the highly variable information contained in the medical records. Categorical variables were compared by use of Fisher’s exact test and chi-square analysis. Continuous variables were compared by use of the Mann-Whitney rank sum test. Kaplan-Meier estimated response curves were compared using the log rank test, and dogs that were still in remission at the time of analysis or lost to follow-up were censored. Simple linear regression was used to evaluate the correlation between quantitative values and PFIs. Data analyses were done using commercial software,^f and P values <0.05 were considered statistically significant. Data are reported as mean±standard deviation (SD), unless otherwise noted.

Results

Seventy-six dogs with histopathologically confirmed multicentric lymphoma were included in this study. Thirty-four dogs were treated with COP, and 42 were treated with COPA chemotherapy. Among the 42 dogs in the COPA group, 18 received one dose of asparaginase and 24 received two doses of asparaginase during induction chemotherapy. This sample size provided 80% statistical power (α=0.05) to detect a 9-week difference in PFI between the two treatment groups. Censored data included one dog in the COP group that was lost to follow-up, one dog in the COPA group that was in remission at the time of analysis, and one dog in the COPA group that was lost to follow-up.

In the COP group, 15 (44.1%) dogs were males, seven of which were castrated, and 19 (55.9%) dogs were females, 13 of which were spayed. There were 11 (32.4%) mixed-breed dogs, five (14.7%) American cocker spaniels, three (8.8%) Doberman pinschers, three (8.8%) golden retrievers, two (5.9%) Labrador retrievers, two (5.9%) miniature schnauzers, and one (2.9%) each of eight other breeds. The mean weight for the COP group was 24.1±12.30 kg, and the mean age was 6.6±2.61 years.

In the COPA group, 23 (54.8%) dogs were males, of which 20 were castrated, and 19 (45.2%) dogs were females, of which 18 were spayed. There were 11 (26.2%) mixed-breed dogs, three (7.1%) Doberman pinschers, three (7.1%) rottweilers, two (4.8%) golden retrievers, two (4.8%) Labrador retrievers, two (4.8%) Pembroke Welsh corgis, and one (2.4%) each of 19 other breeds. The mean weight for the COPA group was 27.0±14.48 kg, and the mean age was 7.3±2.66 years. There were no significant differences in mean age, mean weight, or sex distribution between the groups (P=0.284, P=0.369, and P=0.141, respectively).

In the COP group, eight (23.5%) dogs had stage III lymphoma, seven (20.6%) had stage IV lymphoma, and 19 (55.9%) had stage V lymphoma. Eighteen (52.9%) dogs had no evidence of clinical signs at the time of diagnosis (substage a), and 16 (47.1%) had evidence of clinical signs related to their disease (substage b). In the COPA group, four (9.5%) dogs had stage III lymphoma, 21 (50%) dogs had stage IV lymphoma, and 17 (40.5%) dogs had stage V lymphoma. Twenty-seven (64.3%) dogs were substage a, and 15 (35.7%) dogs were substage b. There were no significant differences in the frequency of clinical stage or substage between the groups (P=0.335, P=0.172, respectively).

At week 2 of COP induction chemotherapy, 18 (53%) dogs were in CR, 15 (44%) dogs had a PR, and one (3.0%) dog had progressive disease. At week 2 of COPA induction chemotherapy, 24 (57.1%) dogs were in CR, 16 (38.1%) dogs had a PR, and two (4.8%) dogs had progressive disease. There was no significant (P=0.470) difference between the groups in the clinical responses at week 2 of induction chemotherapy.

Dogs were evaluated again at week 6 of induction chemotherapy. In the COP group, 24 (70.6%) dogs were in CR, three (8.8%) dogs had a PR, five (14.7%) dogs had progressive disease, and two (5.9%) dogs were euthanized before week 6 because of progressive disease. In the COPA group, 33 (78.6%) dogs were in CR, one (2.4%) dog had a PR, three (7.1%) dogs had progressive disease, four (9.5%) dogs were euthanized before week 6 because of progressive disease, and one (2.4%) dog was lost to follow-up. There was no significant (P=0.887) difference between the groups in the clinical responses at week 6 of induction chemotherapy.

The median initial PFI for dogs treated with COP was 12 weeks (range 4 to 88 weeks), and the median initial PFI for dogs treated with COPA was 25 weeks (range 4 to 52 weeks). However, these PFI values were not significantly (P=0.451) different [Figure 1]. Likewise, there was no significant difference in median initial PFI [Figure 2] between dogs treated with either one (n=18) or two (n=24) doses of asparaginase and those dogs treated with COP alone (n=34; P=0.279, P=0.0858, respectively). The median initial PFI for dogs that received two doses of asparaginase (28 weeks; range 3 to 52 weeks) was significantly (P=0.007) longer than the median initial PFI for dogs given one dose of asparaginase (14 weeks; range 2 to 51 weeks). There were no significant differences (P>0.320 for all comparisons) between median PFI when dogs were grouped by clinical stage, clinical substage, and clinical response at 2 or 6 weeks, in either treatment group. Likewise, age and weight did not significantly (P>0.199 for all comparisons) correlate with PFI in either treatment group.

Discussion

L-asparaginase is combined with other chemotherapeutic drugs and is widely believed to be effective for the treatment of canine lymphoma.^{8,10,12,13,22,23} However, the positive contribution of asparaginase to combination chemotherapy has yet to be determined, because prospective trials studying asparaginase face the obstacles of the drug’s relative expense and inconsistent availability from manufacturers.

The 6-week CR rate (71%) observed in this study for dogs treated with COP was comparable to the 3-week 75% CR rate previously reported.² The 12-week median PFI for dogs treated with COP in this study was considerably shorter than the 24-week and 18-week median PFIs previously reported.^2,8 Differences in PFI determination among studies makes direct comparisons of reported results difficult. For example, in the report of canine lymphoma treatment using similar drugs as in the present study, the duration of remission was defined as the period between initial lymph node reduction and the return of lymphadenopathy to pretreatment size.⁸ In the present study, a patient was considered relapsed at the first sign of lymph node enlargement, which may have occurred long before a return to pretreatment size. Although resulting in a shorter PFI, this more conservative definition provides a critical view of the efficacy of the protocols studied.

Sex, clinical stage, and substage did not affect PFI in either the COP or COPA treatment groups in the present study, although they have been previously associated with prognosis.¹ Unfortunately, because of the retrospective nature of this study and the lack of information available from the medical records, other variables previously reported to have prognostic significance (such as immunophenotype) could not be assessed.

In the study reported here, the addition of asparaginase to the induction phase of a modified COP chemotherapy protocol did not provide additional benefits for the treated dogs. Although dogs treated with two doses of asparaginase during induction chemotherapy had a significantly longer initial PFI than those given one dose of asparaginase, neither asparaginase-treated group had a median initial PFI that was significantly longer than the COP group. Likewise, there were no significant differences in the remission status of the treatment groups at 2 and 6 weeks of treatment, demonstrating that the addition of asparaginase did not increase the percentage of dogs that achieved a CR. In a previous report of a combination chemotherapy protocol using the same drugs as the COPA protocol described here, but with four doses of asparaginase, the overall response rate was 96.7%, which was similar to the 2-week response rate of 95.2% reported in this study.⁸ In the prior study, the median response duration for dogs with lymphoma was 18 weeks, compared to the 14-week (one dose of asparaginase) and 28-week (two doses of asparaginase) median PFIs observed in dogs of the present study. These observations suggested that increasing the number of treatments of asparaginase administered in canine lymphoma chemotherapy protocols may not improve the outcome.

Similar to other COP-type combination protocols, the chemotherapy protocol evaluated in this study was of relatively low dose intensity (i.e., smaller amount of drug delivered per unit of time) and provided shorter PFIs compared to combination chemotherapy protocols for canine lymphoma that incorporate doxorubicin.^6,7,24,25 One study describing the addition of asparaginase to a doxorubicin-based combination chemotherapy protocol for canine lymphoma reported that dogs given asparaginase were three times more likely to respond to treatment, but the use of asparaginase did not significantly influence overall remission and survival times.¹² These findings suggested that asparaginase may have minimal impact on higher dose-intensity combination chemotherapy protocols for canine lymphoma that include doxorubicin.

Conclusion

Based on the results of the present study, asparaginase may be of limited benefit to dogs with multicentric lymphoma when added to the induction phase of a low dose-intensity chemotherapeutic protocol. Because of the expense and inconsistent availability of asparaginase, additional studies are needed to determine its optimal application in the treatment of canine lymphoma.

Acknowledgment

The authors thank Dr. William E. Blevins, Diplomate ACVR, for diagnostic imaging support.