Intermittent Single-Agent Doxorubicin for the Treatment of Canine B-Cell Lymphoma
Canine B-cell lymphoma is a highly treatable disease, but cost and logistical factors may hamper an owner’s ability to pursue treatment of their pet with this disease. The authors evaluated the use of single-agent doxorubicin in an intermittent fashion for efficacy in the treatment of this disease. Morphologic and clinical data were analyzed for prognostic significance. Eighteen dogs with B-cell lymphoma, all with multicentric disease, were enrolled. The overall complete response (CR) rate was 78%, median total doxorubicin remission time (TDR) was 80.5 days, and median overall survival (OS) was 169.5 days. The median number of doxorubicin doses administered was 4.5. First remission times were significantly affected by clinical stage and substage of disease. Outcome for the dogs in this study were similar to those previously reported for single-agent doxorubicin treatment. Additionally, the intermittent nature of the treatments made the described protocol more feasible for the owners who enrolled their pets in this study. Intermittent single-agent doxorubicin is not a substitute for multiagent chemotherapy protocols in the treatment of canine lymphoma; however, it is a reasonable alternative if the cost and time commitments are limiting factors for an owner.
Introduction
Lymphoma is the most frequently diagnosed hematopoietic neoplasm in dogs, with an estimated annual incidence of 13–24 cases/100,000 dogs at risk.1,2 While lymphoma arises from a clonal expansion of lymphoid cells, it is a heterogeneous disease with multiple distinct morphologic and immunophenotypic subtypes. Based on immunophenotype alone, a recent study showed an overall response rate of 100% for dogs with B-cell lymphoma versus 50% for those with T-cell lymphoma when treated with a single dose of doxorubicin.3
Single-agent doxorubicin has been evaluated for treatment of canine lymphoma; however, multiagent chemotherapy is the standard of care for treatment of non-Hodgkin’s-like lymphoma in dogs. The most frequently used multiagent protocols include cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP-based) protocol and may also include l-asparaginase. Treatments in those protocols occur on a regular basis, from weekly to monthly, for up to 2 yr based on the protocol used. One widely used CHOP-based protocol includes 16 treatments over 24 wks.4 While efficacious in obtaining remission of lymphoma in the majority of patients, the remission and survival times for single-agent doxorubicin are shorter than those for CHOP-based protocols. Response rates reported for canine lymphoma treated with single-agent doxorubicin range from 69% to 74%, and median remission and survival times ranged from 131 days to 206 days and from 169.5 days to 270 days, respectively.5–8
For some owners, weekly visits to a veterinarian are not possible either due to perceived stress on the pet; conflicts due to work; personal scheduling issues; or cost of the multiple, frequent visits. Additionally, not all owners willing to pursue treatment of lymphoma live in an area with a veterinarian equipped to administer chemotherapy to pets. In the authors’ practices, clients drive an average of 100 miles in each direction to seek oncologic care, making frequent visits a significant challenge to owners. Many owners are willing to undergo treatment of lymphoma for their dog if they are likely to have a prolonged, high-quality life and the budget and time commitment fit their lifestyle. Should an owner decide not to pursue treatment of their dog with lymphoma due to cost, stress, or time commitment issues, there is potential lost income for the veterinary practice. Treatment options considered less intense, yet likely to extend high quality life, would be attractive to both pet owners and veterinarians.
In an attempt to decrease perceived stress, cost, and owner time commitment in the treatment of lymphoma, the authors elected to evaluate the response of canine B-cell lymphoma to single-agent doxorubicin treatment given on an intermittent basis. The authors hypothesized that there would be similar response rates, remission durations, and survival times as those previously published for traditional single-agent doxorubicin treatment protocols. Additionally, the authors evaluated morphologic and clinical characteristics of each patient as prognostic indicators.
Materials and Methods
Study Population
Dogs with B-cell lymphoma were enrolled in this prospective study at the Kansas State University Veterinary Medical Teaching Hospital from 2008 to 2010. Enrollment criteria included a diagnosis of B-cell lymphoma with measurable affected lymph nodes; no prior chemotherapy (prednisone therapy for ≤ 7 days was acceptable); acceptable candidate for doxorubicin therapy on echocardiographic examination (based on fractional shortening, ejection fraction, and chamber dimensions); willingness of the client for all chemotherapy treatments during the study period to be performed at the study institution; and informed, written client consent with the right to withdraw from the study at any point in time. A microscopic diagnosis of lymphoma for study enrollment was established by either histopathology or cytology of an affected lymph node sample. B cell phenotype was confirmed either by immunohistochemistry of histopathology specimens or by flow cytometric immunophenotyping of a lymph node aspirate if histopathology specimens were not available prior to study enrollment. This study was approved by the Kansas State University Animal Care and Use Committee.
Patient Evaluation
At the time of study enrollment, all patients were evaluated with a physical examination, complete blood cell count (CBC), serum biochemical profile, urinalysis, thoracic radiographs, abdominal ultrasound, bone marrow aspirate, and echocardiogram. Additionally, a representative peripheral lymph node was either completely excised or biopsied using a biopsy gun with a 14-gauge disposable tissue biopsy needlea. Histopathologic samples were evaluated and World Health Organization (WHO) classification of lymphoma was performed by one pathologist (J.C.N.).9 Immunohistochemistry of all histologic specimens was performed with antibodies for CD3b and CD79ac using standard techniques. A representative lymph node was also aspirated and samples submitted in 5 mL 0.9% NaCld for immunophenotyping by flow cytometry. Flow cytometry was performed as previously described.10 The panel of antibodies applied to all samples for cytometric evaluation to confirm a B-cell immunophenotype included CD45e (leukocyte antigen), CD21f, immunoglobulin Mg, immunoglobulin Gh, CD79ac (B cell antigens), and CD4i and CD5j (T cell antigens). Isotype controls were used for all antibodies.
Response Evaluation
Response to therapy was determined at each evaluation, and either lymph node assessments or cytology findings were compared with the most recent assessment. Response to therapy was defined as complete response (CR, a 100% reduction in lymph node size as assessed by lymph node palpation), a partial response ([PR], < 100% but > 50% regression of lymph node size), stable disease ([SD], < 50% regression of lymph node size or < 50% increase in lymph node size), or progressive disease ([PD], > 50% increase in lymph node size). If response to therapy was in question, a lymph node aspirate and cytology was performed to evaluate for microscopic evidence of lymphoma in the lymph node.
Treatment Protocol
The induction phase of the treatment plan included one doxorubicin treatment q 2 wk for three treatments (days 0, 14, and 28). Doxorubicin was prescribed at a standard dose of 30 mg/m2 (or 1 mg/kg for dogs weighing < 15 kg). Dogs were evaluated by physical examination and CBC 1 wk following each doxorubicin treatment. During the induction phase of the protocol, dogs were treated with a minimum of two doses of doxorubicin before their response to therapy was definitively assessed. If a CR was detected, a third dose of doxorubicin was administered to complete the induction phase of the protocol. If a CR was not present after two doses of doxorubicin, they could be taken off study and additional treatments pursued. For dogs achieving a CR and completing the initial three doxorubicin treatments, they were evaluated for lymph node enlargement by their veterinarian either bimonthly or sooner if deemed necessary by the owner. One dose of doxorubicin was administered at the previously stated dose if the dog was confirmed to have PD. This intermittent doxorubicin administration was performed at each PD based on lymph node cytology until the lymphoma was resistant to doxorubicin therapy based on lack of lymph node regression following treatment. An echocardiogram was performed prior to treatments three, six, and eight if the dog continued to respond to doxorubicin. When the lymphoma became resistant to doxorubicin treatment, the dog was considered off study and the owner was free to choose additional therapy as they so desired.
Statistical Analysis
End points evaluated included first duration of remission ([DOR], first remission following the induction phase of treatment), total doxorubicin remission ([TDR], total time responsive to doxorubicin), and overall survival (OS). End points were calculated from the date of first doxorubicin administration. Independent group comparisons (t-test or analysis of variance) were made in days between first DOR, TDR, or OS and the categorical data of number of doxorubicin treatments, response to treatment (CR versus PR/SD/PD), WHO classification, clinical stage, clinical substage, and the presence of anemia or hypoalbuminemia. Where appropriate, a Newman-Keuls test was applied to determine individual differences post hoc. Independent group comparisons were also made to compare the clinical response (CR versus PR, SD, and PD) to the number of days between treatments three and four and four and five. A χ2 analysis was performed to compare clinical response with the variables of WHO classification, clinical stage, clinical substage, and the presence of either anemia or hypoalbuminemia. Simple linear regression was performed to compare the first DOR, TDR, or OS in days with the number of days between individual treatments (for treatments one through six). Independent group t test was used to compare the number of doxorubicin treatments with the first DOR, TDR, and OS. Survival analysis based on stage and substage parameters was performed using the Kaplan-Meier method. Dogs that survived were censored at the time of data analysis. A P ≤ 0.05 was considered significant for all results, and all data were analyzed using a commercially available software programl.
Results
Eighteen client-owned adult dogs with naturally occurring B-cell lymphoma were enrolled in this prospective study. Breeds represented are included in Table 1. Mean and median age of the enrolled patients was 8.57 yr and 7.95 yr, respectively (range, 3.2–14.1 yr). There were 10 castrated males, 2 males, and 6 spayed females in the study population. All dogs had multicentric disease. Lymphoma stage and substage included stage III (n = 3), stage IV (n = 6), stage V (n = 9), substage a (n = 12), and substage b (n = 6). All patients with stage V disease had evidence of bone marrow infiltration based on cytology of a bone marrow aspirate. Two of the dogs with stage V disease also had evidence of pleural effusion, 1 of which had a bronchial pulmonary pattern on thoracic radiographs. Three dogs were stage IIIa, 5 dogs were stage IVa, 1 dog was stage IVb, 4 dogs were stage Va, and 5 dogs were stage Vb. On CBC and serum biochemical profile, 4 dogs were anemic and 4 dogs were hypoalbuminemic at study enrollment. No patient was hypercalcemic. All patients were confirmed with B-cell lymphoma using either immunohistochemistry (n = 3), flow cytometric immunophenotyping (n = 3), or both (n = 12). Fifteen of 18 patients had histologic specimens available for WHO classification as summarized in Table 1.
Centro, centroblastic; DLBCL, diffuse large B-cell lymphoma; immuno, immunoblastic; NA, not available; OS, overall survival; TDR, total doxorubicin remission time; WHO, World Health Organization.
Doxorubicin was administered at 30 mg/m2 in 17 of 18 patients and 1 mg/kg was administered to 1 patient weighing 6.6 kg. The number of doxorubicin doses/patient include one (n = 3), two (n = 1), three (n = 3), four (n = 5), five (n = 5), and nine (n = 1). The mean and median number of doxorubicin doses administered was 3.8 and 4.5, respectively, (range, 1–9). The patient that received nine doses of doxorubicin was dosed at 1 mg/kg. His calculated cumulative doxorubicin dose was 170.28 mg/m2.
Fourteen of 18 dogs achieved a CR for an overall response rate of 78%. However, 1 dog relapsed between treatments two and three and did not respond to the third treatment. Another dog had a CR to doxorubicin but died due to clinical signs consistent with doxorubicin toxicity (both hematologic and gastrointestinal) 10 days following the first doxorubicin treatment. Therefore, 12 of 18 dogs (67%) were in CR at the conclusion of the induction phase of the protocol. Clinical response was significantly associated with disease substage based on χ2 analysis evaluating categorical data. Dogs with substage a disease were significantly more likely to obtain a CR with doxorubicin treatment than dogs with substage b (P = 0.002).
The median first DOR was 68.5 days (range, 0–335 days). The median TDR was 80.5 days (range, 0–335 days) and the median OS was 169.5 days (range, 0–865 days). Two dogs were alive and in CR at 335 days and 865 days, respectively. Those dogs were in remission following three and five doses of doxorubicin, respectively. First DOR was significantly affected by clinical stage of disease. Dogs with stage V disease had a significantly shorter first DOR (median, 41 days; mean, 37.89 days) than those with stage III (median, 216.67 days; mean, 249 days) or stage IV (median, 119.5; mean, 121.67 days) disease (P = 0.0051; Figure 1). First DOR was also significantly affected by clinical substage of disease. Substage b (median, 0 days; mean, 22.17 days) had a significantly shorter first DOR than substage a (median, 89 days; mean, 138.36 days; P = 0.005; Figure 2). Dogs with a CR had a significantly longer first DOR (P = 0.023) and OS (P = 0.039) than those with PR, SD, or PD. Neither the presence of anemia or hypoalbuminemia at diagnosis nor the morphologic classification was significant for response to treatment, first DOR, TDR, or OS.
Citation: Journal of the American Animal Hospital Association 49, 6; 10.5326/JAAHA-MS-5929
Citation: Journal of the American Animal Hospital Association 49, 6; 10.5326/JAAHA-MS-5929
When resistant to doxorubicin treatment, nine dogs received rescue therapies and three did not. One dog was withdrawn from the study in CR following the fourth doxorubicin treatment due to owner preference. Three dogs died prior to receiving rescue therapies, and two were alive and in CR from doxorubicin treatment at the time this manuscript was prepared (one after receiving five doses of doxorubicin and one after the induction phase of the protocol). Rescue therapies used include l-asparaginase; prednisone; cyclophosphamide, vincristine, and prednisone; and lomustine therapy. Because rescue therapy was not standardized and not all were performed at the authors’ institution, response could not be evaluated.
Discussion
Doxorubicin has been shown to be an effective drug for the treatment of canine lymphoma. Typically it has been given q 3 wk for five treatments when used as a single agent in the treatment of canine lymphoma. Response rates reported for single-agent doxorubicin range from 69% to 74% and median remission and survival times range from 131 days to 206 days and 169.5 days to 270 days, respectively.5–8 In the study presented here, the authors found an acceptable overall response rate of 78%, an overall median TDR of 80.5 days, and a median OS of 169.5 days. When considering the 12 patients that achieved CR following the induction phase of the protocol, the median TDR was 157.5 days and the median OS for those patients was 273.5 days, with a median of 4.5 doxorubicin treatments. Those results are comparable with what has previously been reported for single-agent doxorubicin therapy.
Although the intent of this study was for the dogs to receive doxorubicin until they became resistant, one dog was withdrawn from the study prior to exhibiting doxorubicin resistance, another died at home with symptoms of doxorubicin toxicity, and two dogs remained alive and in CR at the time this manuscript was prepared. Therefore, 25% of the dogs with a CR did not have doxorubicin resistance. Additionally, five dogs with CR survived beyond 300 days (301 days, 335 days, 440 days, 465 days, and 865 days). The two dogs alive and in CR remained so at 335 days and 865 days (at the time this manuscript was prepared), receiving three doses and five doses of doxorubicin. It was unknown whether those dogs would have experienced similar remission and survival times had they received doxorubicin q 3 wk for five treatments versus the protocol described herein.
With a median of 4.5 doxorubicin treatments, the authors feel the protocol described herein demonstrated reasonable results, particularly for clients interested in a relatively low cost, low time commitment protocol for their pet. The intermittent doxorubicin protocol should not be substituted for standard CHOP-based protocols for an owner who is interested in a more dose intense protocol. However, the time and cost associated with 16 treatments over 24 wk time as necessary in one CHOP-based protocol may not be feasible for some owners.4 With the exception of one dog that died due to gastrointestinal and hematologic toxicities of doxorubicin, the protocol described herein was very well tolerated, with no other dog requiring either hospitalization or supportive care while receiving the doxorubicin. The breed of the dog that died of presumed doxorubicin toxicity was a border collie. Why that patient had such a severe reaction is unknown. Less than 5% of border collies are reportedly affected with a multidrug resistance 1 (MDR1) mutation that would make them more sensitive to the toxicities of MDR1 substrate drugs (such as doxorubicin); however, it is possible the border collie described in this report had an MDR1 mutation leading to the severe toxicity encountered.11 It is also possible that patient had either another unknown genetic predisposition to toxicity with doxorubicin or that another illness or toxicity exacerbated his gastrointestinal signs.
Multiple classification systems have been described to differentiate lymphomas. The most recently accepted system in veterinary medicine is based on the revised WHO system of classification described for human hematologic malignancies.9 That system considers cellular morphology, immunophenotype, and genetic features as well as clinical syndromes when classifying lymphoma. In the cases reported herein, the authors did not find that lymphoma type based on the WHO classification system was of prognostic significance. Additionally, the authors did not find that previously noted negative prognostic indicators such as either anemia or hypoalbuminemia were associated with prognosis. Most likely, the lack of prognostic significance for those variables was due to the small number of cases and lack of power in this study.
Conclusion
Overall, intermittent single-agent doxorubicin appears to be a well-tolerated, less intense protocol for canine B-cell lymphoma. While intermittent single-agent doxorubicin is not a substitute for multiagent chemotherapy protocols in the treatment of canine lymphoma, it is a reasonable alternative if cost and time commitments are limiting factors for an owner.
Kaplan-Meier curve for 18 dogs treated with intermittent single-agent doxorubicin for B-cell lymphoma. First remission times were significantly affected by clinical stage of disease. Patients with stage V disease had a significantly shorter first remission (median, 41 days) than those with either stage III (median, 216.67 days) or stage IV (median, 119.5 days) disease (P = 0.0051). The V denotes censored patients at the time of analysis.
Kaplan-Meier curve for 18 dogs treated with intermittent single-agent doxorubicin for B-cell lymphoma. First remission times were significantly affected by clinical substage of disease. Substage b (median, 0 days) patients had a significantly shorter first remission time than substage a patients (median, 89 days; P = 0.005). The V denotes censored patients at the time of analysis.
Contributor Notes
K. Reeds’ updated credentials since article acceptance are MS, DVM, DACVIM.
K. Reeds’ present affiliation is Department of Veterinary Clinical Sciences, Oklahoma State University, Stillwater, OK.
D. Burr’s updated credentials since article acceptance are MS, DVM, DACVIM.
D. Burr’s present affiliation is Carolina Veterinary Specialists, Greensboro, NC.
