Introduction

Lymphoma is one of the most common neoplasms in the dog, accounting for 7–24% of all canine neoplasms and 83% of canine hematopoietic malignancies.^1,2 Combination chemotherapy is the treatment of choice for dogs with lymphoma (LSA), resulting in a high response rate of approximately 70–90%, but the cure rate is low.^3–11 Cellular resistance to chemotherapy, or inadequate dosing and frequency of administration of chemotherapy, are the usual cause of relapse.¹² Rescue chemotherapy protocols are intended to re-establish remission in dogs who have relapsed or to induce remission in dogs who are resistant to first-line chemotherapy protocols (refractory lymphoma). There are many rescue chemotherapy protocols described in the veterinary literature, with response rates ranging from 28 to 87% and a median duration of response of 1–5 mo.^13–26

Cytosine arabinoside (cytarabine) is an antimetabolite chemotherapy drug that has activity against rapidly dividing cells.^27,28 It acts as an analogue of deoxycytidine and has multiple effects on DNA synthesis.²⁷ The mechanism of action of cytarabine involves the inhibition of DNA polymerase α, incorporation into DNA, and termination of DNA chain elongation, leading to the inhibition of the function of the DNA template and subsequent synthesis.²⁷ Cytarabine is commonly used in the treatment of acute and chronic myelogenous leukemia, non-Hodgkin’s LSA, and acute lymphoblastic leukemia in human oncology.²⁸ It is often incorporated into treatment protocols for human patients diagnosed with lymphoma involving the central nervous system because of its capacity to cross the blood–brain barrier.²⁹ Bleomycin is an antibiotic antineoplastic agent that was first isolated from glycopeptides of the culture broths of the fungus Streptomyces verticillus.³⁰ The cytotoxicity and antitumor activity of bleomycin is associated with a direct DNA damage.³¹ After bleomycin binds to DNA, single-strand and double-strand breaks are produced due to the formation of an activated oxygen complex, leading to cell death.³¹ Bleomycin has attracted great interest due to its tendency to concentrate in lymphoid tissues and the drug’s lack of myelosuppression.^32–34 It was shown to have important activity in human cancers, such as Hodgkin’s and non-Hodgkin’s LSA, testicular cancer, malignant pleural effusions, cervical and penile cancers, and head and neck cancers.^33,35–38 Third-line chemotherapy protocols used to treat relapsed LSA in human oncology often include the administration of both cytarabine and bleomycin because these agents are not typically incorporated into frontline chemotherapy regimens.^39,40

In veterinary medicine, cytarabine is used as part of the COAP and DMAC chemotherapy protocols for dogs diagnosed with LSA to induce remission or as rescue therapy, respectively.^41–43 This drug has been evaluated as a first-line single agent for dogs with LSA, but no objective response was noted.⁴⁴ Marconato et al. published a report that indicated that the addition of cytarabine to a CHOP-based protocol was beneficial for treatment of canine LSA cases that have bone marrow involvement.⁴⁵ Bleomycin is administered intralesionally to dogs with acanthomatous ameloblastoma.⁴⁶ Newer reports utilized bleomycin as part of electrochemotherapy for dogs with mast cell tumor, melanoma, transmissible venereal tumor, fibrosarcoma, histiocytic sarcoma, and squamous cell carcinoma.^47–49

The rationale approach for considering Bleo/Cytarabine as a potential rescue protocol involved the use of non-cross resistant drugs to which patients were not previously exposed because dogs with relapsed lymphoma usually have tumor clones or tumor stem cells that are more resistant to chemotherapy than the original tumor.⁵⁰ Furthermore, the authors also speculated that combining bleomycin, a nonmyelosuppressive drug, with cytarabine, a more aggressive agent that acts against neoplasms with a rapid growth rate with cells passing from G1 to S-phase, could overcome inherent drug resistance by the tumor with tolerable side effects.^27,32–34

The purpose of the study reported here is to evaluate the efficacy and toxicity of a bleomycin and cytarabine combination protocol for dogs with LSA that have developed resistance or that failed to respond to previously administered chemotherapy.

Materials and Methods

The medical records of dogs diagnosed with relapsed LSA treated with the Bleo/Cytarabine protocol at Michigan State University-Veterinary Teaching Hospital and Pittsburgh Veterinary Specialty and Emergency Center between 2004 and 2011 were retrospectively reviewed. Owner informed consent was obtained before treatment with Bleo/Cytarabine. Dogs were eligible for inclusion in the study if they met the following criteria: (1) had cytologic or histologic confirmed high-grade LSA; (2) had prior induction with a doxorubicin-based chemotherapy protocol; and (3) had measurable, relapsed LSA. Exclusion criteria included patients with insufficient follow-up in the medical record and patients initiating corticosteroids at the same time as Bleo/Cytarabine. Patients receiving corticosteroids from the time of at least their immediate previous chemotherapy protocol were not excluded.

Patient data evaluated for analysis included signalment; stage; substage; immunophenotype; type and number of previous chemotherapy treatments; presence of concurrent diseases; dose, number, and frequency of Bleo/Cytarabine cycles; concurrent medications while on Bleo/Cytarabine therapy; and Bleo/Cytarabine toxicity, response, and duration of response.

In all dogs, a complete blood count and complete physical exam, including tumor volume measurements with calipers, were performed on each day of bleomycin administration and 7 days after the second dose of bleomycin of each cycle.

The Bleo/Cytarabine protocol consisted of bleomycin given subcutaneously at 15 mg/m² on days 1 and 8. Cytarabine was administered subcutaneously at 100 mg/m², with the total dose divided every 8 hr, on days 1–5 of a 21-day cycle (Table 1). Cytarabine was dispensed to owners to be administered at home. Owners were instructed to wear latex gloves when handling the drug. Treatments were continued for as long as the dogs had an objective response (complete response, partial response, stable disease) and showed no signs of drug toxicity. Toxicosis associated with Bleo/Cytarabine therapy was monitored by the evaluation of hematologic data, physical examination, and history obtained from the owners. Toxicity was graded according to the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events criteria.⁵¹

TABLE 1 Bleo/Cytarabine Chemotherapy Protocol for Treatment of Dogs with Relapsed Lymphoma

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Disease response was determined according to the response evaluation criteria for peripheral nodal lymphoma in dogs (v1.0)—a Veterinary Cooperative Oncology Group consensus document. ⁵² Peripheral lymph node measurements were determined by physical examination using calipers immediately before the first dose of Bleo/Cytarabine, and tumor response was determined each time dogs were examined. A complete response was defined as disappearance of all measurable disease. All lymph nodes must be nonpathologic in size in the judgment of the evaluators. A partial response was defined as at least 30% decrease in the mean sum of the longest diameter of peripheral lymph nodes. Progressive disease was defined as at least 20% increase in the mean sum of the longest diameter of peripheral lymph nodes. Stable disease was defined as neither sufficient decrease to qualify for partial response nor sufficient increase to qualify for progressive disease.

First-line chemotherapy protocol time to progression (TTP) was defined as the time from diagnosis to the time of progression of disease. Bleo/Cytarabine TTP was defined as the time from date of first Bleo/Cytarabine treatment to the time of disease progression. For the purposes of calculations, dogs were considered lost to follow-up and were censored when they did not return for scheduled recheck appointments and the referring veterinarian or owner could not be contacted. Time to tumor progression estimates were generated by the Kaplan-Meier method. Log-rank test and Cox regression were used for univariate and multivariate analysis of potential risk factors, respectively. The risk factors included clinical stage, substage, and immunophenotype; number of prior chemotherapy drugs; and number of chemotherapy protocols before the Bleo/Cytarabine rescue protocol was administered. A P value of <.05 was considered to be statistically significant. Commercially available software was used for all statistical calculations^a.

Results

Patients

A total of 20 dogs were included in this retrospective study. Seventeen dogs were treated at Michigan State University-Veterinary Teaching Hospital and three dogs were treated at Pittsburgh Veterinary Specialty and Emergency Center. The breeds that were represented included mixed breed dogs (eight), golden retrievers (two), boxers (two), and Labrador retrievers (two). The remaining six dogs consisted of one of each of the following breeds: Australian shepherd, rottweiler, miniature schnauzer, Pit bull terrier, beagle, and Yorkshire terrier. The median age at the time of diagnosis was 7 yr (range 4–12 yr) and the median weight was 33.6 kg (range 4–64.5 kg). Seven dogs were spayed females and 13 were neutered male dogs. The characteristics of Bleo/Cytarabine-treated dogs, including initial disease stage, substage, and immunophenotype, are summarized in Table 2.

TABLE 2 Characteristics of 20 Dogs with Relapsed Lymphoma Treated with Bleo/Cytarabine Protocol

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All dogs had staging tests prior to induction therapy that included lymph node cytology, minimum database (complete blood count, biochemistry profile, urinalysis), thoracic radiographs, and abdominal ultrasound. Bone marrow aspirates (4 of 20), lymph node biopsy (9 of 20), and immunophenotyping (12 of 20) were not routinely done.

The median number of chemotherapy drugs received per case before initiating the Bleo/Cytarabine protocol was 8.5 (range 5–11). Dogs received three to seven rescue protocols before initiating Bleo/Cytarabine. The median number of rescue protocols per dog was five. Prior frontline chemotherapy protocols and rescue regiments to Bleo/Cytarabine included the following: UW-25 (19)⁵³; l-asparaginase (20)^20,26; CCNU (12)¹⁵; actinomycin-D and dacarbazine (5)^17,23; doxorubicin and dacarbazine (5)^17,23; doxorubicin and temozolamide (5)²³; MOPP (12)¹³; doxorubicin (2)¹⁶; single-agent cytosine arabinoside (2)⁴⁴; single-agent bleomycin (1)⁵⁴; and single-agent cladribine (1; cladribine was administered subcutaneously at 0.05 mg/kg/day, with the total dose divided q 8 hr, on days 1–5, of a 14-day cycle). The median first-line chemotherapy protocol TTP was 111 days (range 21–895 days; Figure 1).

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Bleo/Cytarabine Administration and Evaluation of Tumor Response

A total of 23 cycles of Bleo/Cytarabine were administered. Each dog received a median of one cycle (range one to two cycles). Nine of twenty dogs did not complete the full cycle.

Nineteen of the twenty dogs could be evaluated for response to Bleo/Cytarabine treatment. One dog was censored from the response analysis due to being lost to follow-up for terminal staging. Response assessment revealed an overall response rate of 36.8%. Seven dogs had a partial response, three dogs had stable disease, and nine had progressive disease while on Bleo/Cytarabine therapy. The median duration of response for the dogs who achieved a partial response was 15 days (range 13–21 days; Figure 2). No risk factors for response to treatment were identified in our study population.

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Four of the Bleo/Cytarabine responders were treated concurrently with corticosteroids. Corticosteroids were not initiated at the same time as Bleo/Cytarabine, and all of these dogs had received corticosteroids from the time of at least their immediate previous chemotherapy protocol.

Bleo/Cytarabine Toxicity

Twenty dogs could be evaluated for Bleo/Cytarabine toxicity. Bleo/Cytarabine was generally well tolerated, although hematological and gastrointestinal toxicities were observed (Table 3). Thrombocytopenia was the most common toxicity noted. Of the eight dogs that developed thrombocytopenia, five had a grade 2 and three had a grade 3. Dogs were not thrombocytopenic prior to the administration of Bleo/Cytarabine. Other hematologic toxicities included two grade 2 and one grade 4 neutropenia and two grade 2 anemia. Of the three dogs that developed gastrointestinal toxicity, one had grade 2 vomiting and two had grade 1 diarrhea.

TABLE 3 Toxicosis Associated with Bleo/Cytarabine Therapy for Dogs with Relapsed Lymphoma According to the VCOG-CTCAE Criteria

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Discussion

Our study demonstrates that Bleo/Cytarabine therapy was overall well tolerated with mild to moderate reversible toxicities in some cases. Moderate to severe thrombocytopenia was the most common hematologic toxicosis and was identified in 40% (8 of 20) of dogs. None of the dogs had spontaneous bleeding or required hospitalization due to Bleo/Cytarabine adverse effects. It is possible that the thrombocytopenia noted in these patients may have been associated with cumulative bone marrow toxicosis induced by prior chemotherapy. Furthermore, thrombocytopenia may also have been induced by the infiltration of malignant cells into the bone marrow.

Results of the present study suggested that the combination of bleomycin and cytosine arabinoside had minimal activity when used as a rescue therapy for heavily pretreated canine lymphoma. The overall response rate was 36.8%, and none of the dogs achieved a complete response. Additionally, the median TTP associated with Bleo/Cytarabine administration was only 15 days. The low response rate and short duration of response of this study may have been the result of acquired multidrug chemotherapy resistance, because the majority of our dogs had received several other rescue protocols prior to the administration of Bleo/Cytarabine. None of the dogs received Bleo/Cytarabine as their first rescue treatment. A better response rate or more durable responses may have been achieved if this protocol was initiated in a different setting, such as a first-line rescue protocol.

Our report suffers from several limitations, including the retrospective nature, small sample size, and lack of complete staging at the beginning of Bleo/Cytarabine treatment and at the time of disease progression. Additionally, bone marrow aspirates were not performed in most dogs prior to the initiation of chemotherapy and after the development of thrombocytopenia. Therefore, the cause of thrombocytopenia in these patients could not be fully determined.

Conclusion

This study demonstrates that the chemotherapy protocol Bleo/Cytarabine has limited value for heavily pretreated dogs with relapsed lymphoma. Although thrombocytopenia was a common toxicosis, in general, the protocol was well tolerated. Future studies should be designed to evaluate Bleo/Cytarabine as a first-line rescue therapy.