Retrospective Evaluation of Melphalan, Vincristine, and Cytarabine Chemotherapy for the Treatment of Relapsed Canine Lymphoma
ABSTRACT
Dogs diagnosed with multicentric lymphoma often relapse following induction therapy within the first year of treatment. The primary aim of this study was to evaluate the tolerability of a novel drug combination using melphalan, vincristine, and cytarabine (MOC) for the treatment of relapsed lymphoma. On day 1, dogs were treated with vincristine (0.5–0.6 mg/m2 IV) and cytarabine (300 mg/m2 IV over 4–6 hr or subcutaneously over 2 days). On day 7, dogs were treated with melphalan (20 mg/m2per os). This 2 wk protocol was repeated for at least three cycles or until treatment failure. Twenty-six dogs were treated with MOC and met the inclusion criteria. Twenty-three dogs had toxicity data, and all experienced adverse events with the majority graded as mild. The overall response rate was 38%, which included 19% of dogs who achieved a complete response. The median progression-free survival was 29 days (range 1–280 days). The overall clinical benefit was 65% for a median of 37 days (range 33–280 days). MOC is a safe treatment option for relapsed lymphoma in dogs.
Introduction
Canine high-grade lymphoma initially responds well to multiagent chemotherapy, with 80–90% of dogs experiencing a complete response.1,2 These responses are unfortunately short-lived, and relapse often occurs within the first year. Although second remissions may be attained with rescue chemotherapy, the response rates and durations are generally inferior compared with first-line therapy. Numerous rescue protocols have been investigated previously with reported response rates ranging from 7 to 74% and response durations ranging from 1 to 5 mo.3–18 Chemotherapy-resistant lymphoma remains clinically challenging to treat, and thus, continued investigation into novel protocols is warranted.
Combination chemotherapy, especially for the treatment of lymphoma, may be considered advantageous compared with single-agent protocols.19–21 This is likely due to combination protocols using multiple mechanisms to target cancer cells while slowing the development of cellular drug resistance. Ideal multiagent protocols combine drugs with known single-agent efficacy and minimal overlapping toxicities.22
Vincristine and melphalan have demonstrated efficacy both as single agents and as part of combination protocols for the treatment of canine lymphoma.2–7,23 Although cytarabine has not been found to be efficacious as a single agent for the treatment of canine lymphoma, it has been reported as efficacious as part of combination protocols.2,3,6,10,24,25 Additionally, a prospective study of dogs with treatment-naive stage V lymphoma reported the addition of cytarabine into a vincristine, cyclophosphamide, doxorubicin, and prednisone (CHOP)–based protocol generated significantly higher response rates and longer survivals.26
Furthermore, these chemotherapeutics have disparate mechanisms of action. Vincristine is an antimicrotubule agent that binds tubulin, inhibiting microtubule assembly resulting in metaphase arrest and cytotoxicity.27 Melphalan is a cell-cycle nonspecific, bifunctional alkylating agent within the nitrogen mustard subclass.28 Cytarabine is an antimetabolite that acts as an analog of deoxycytidine. Intracellular phosphorylation generates arabinosylcytosine triphosphate, which competitively inhibits DNA polymerase α.29 Incorporation of cytarabine into DNA leads to cytotoxicity during the S phase of the cell cycle.30 The dose-limiting toxicity for cytarabine and melphalan is thrombocytopenia, whereas the dose-limiting toxicity of vincristine is neutropenia or gastrointestinal toxicity.3,6,7,23,24 Chemotherapeutics frequently have overlapping toxicities, and when used in combination, the dosing schedule may be altered to minimize additive effects.31
The primary aim of this study was to retrospectively evaluate the adverse event (AE) profile of combination chemotherapy with melphalan, vincristine, and cytarabine (MOC) for the treatment of relapsed canine multicentric lymphoma. A secondary aim was to evaluate the efficacy of this novel drug combination.
Materials and Methods
Data were obtained retrospectively from dogs with relapsed or refractory multicentric lymphoma intending to treat with MOC at Oregon State University from March 2013 and March 2020. Informed consent was obtained from all owners, and all dogs were clinically managed according to contemporary standards of care. Dogs were included if they had a cytologic or histologic diagnosis of high-grade or large cell lymphoma by a board-certified veterinary pathologist, had previously been treated with chemotherapy for lymphoma, and had measurable disease at the time of treatment initiation. Data collected included signalment, clinical stage at diagnosis, substage at diagnosis, immunophenotype, previous chemotherapy treatment protocols, and response, date of relapse(s), method of determining relapse, total cycles of MOC received, adverse events, date of progression after MOC, additional treatment protocols and response, date of death, and cause of death.
Dogs who lacked complete disease staging (thoracic radiographs, abdominal ultrasound, cytology of other organs/bone marrow) were included in the study. When performed, stage and substage were assigned at initial diagnosis and relapse according to the modified World Health Organization classification for canine lymphoma.32 Similarly, although immunophenotype was not required for inclusion in this study, when performed via immunocytochemistry, flow cytometry, or polymerase chain reaction for antigen receptor rearrangement, results were recorded.
Treatment Protocol
The dosages used in the treatment protocol were determined based on previously published multiagent chemotherapy protocols for the treatment of canine lymphoma.2,3,6,7,10,24 The treatment protocol is summarized in Table 1. Briefly, dogs were treated with vincristine and cytarabine on day 1. Cytarabine was administered either as a continuous IV infusion over 4–6 hr immediately following IV vincristine or subcutaneously over 2 days. On day 7, dogs were treated with melphalan. Treatment order could be altered depending on clinician or owner preference, e.g., melphalan administered during the first week and vincristine and cytarabine administered during week 2. Dogs were variably prescribed a steroid (prednisone or dexamethasone) throughout the treatment protocol depending on clinician preference. The cycle was repeated for at least three cycles or until treatment failure was documented. Antiemetics and antidiarrheals were prescribed prophylactically or symptomatically depending on clinician preference.
Response to the therapy was evaluated through physical examinations weekly or biweekly depending on owner and clinician preference. Dogs with inadequate follow-up were excluded from the response assessment. Adequate follow-up was defined as a physical examination at least 1 day after treatment. Response was determined using the Veterinary Cooperative Oncology Group (VCOG) response evaluation criteria for lymphoma.33 A complete response (CR) was defined as resolution of all measurable peripheral lymphadenopathy. A partial response (PR) was defined as ≥30% reduction in the sum of the longest diameters of measurable peripheral lymph nodes. Stable disease (SD) was defined as <30% reduction or <20% increase in sum of the longest diameters of measurable peripheral lymph nodes. Progressive disease (PD) was defined as >20% increase in the sum of the longest diameter of measurable peripheral lymph nodes. Clinical benefit was defined as CR, PR, or SD. All responses, including SD, had to be maintained for ≥14 days to be classified as such. Evidence of gastrointestinal and other toxicities was assessed by evaluation of the medical records, including owner-reported history and physical examination findings. Hematological toxicities were evaluated every 7–10 days, and physical examinations were performed weekly or biweekly depending on owner preference. Dogs with inadequate follow-up were excluded from toxicity assessment. A complete blood count 1 wk after treatment and a physical examination 1–2 wk after treatment was defined as adequate follow-up to assess toxicity. Adverse events were graded at the time of patient assessment and retrospectively according to the VCOG Common Terminology Criteria for Adverse Events v2.34 Dose adjustments and treatment delays were permitted based on owner and clinician discretion, commonly if VCOG grade >2 toxicity occurred. If dogs had pre-existing cytopenias, hematological toxicity was attributed to MOC and included in analysis only if the grade was further increased from pretreatment levels.
Statistics
Continuous data were expressed as medians and ranges, and categorical data were expressed as frequencies and percentages. The primary endpoint was progression-free survival (PFS), which was defined as the date of MOC treatment initiation to the date of PD or date of death from any cause. If PD was not documented by a veterinarian, dogs were censored from PFS analysis from the date of the last physical examination. Data were tested for normality using Pearson’s correlation coefficient. Nonparametric data were compared using a Wilcoxon rank-sum test. Overall survival was not evaluated due to the variable pursuit of other rescue protocols. The Kaplan-Meier method was used to estimate and display the distribution of PFS. Differences between groups were compared using log-rank analysis. Variables with values of P ≤ .05 were considered significant. All statistical analyses were performed using standard statistical softwarea.
Results
Case Demographics
Twenty-six dogs met the inclusion criteria and were treated with MOC at Oregon State University between March 2013 and March 2020. Three dogs were excluded from the toxicity analysis due to inadequate follow-up (Figure 1). Two dogs were excluded from PFS analysis. Both dogs had experienced a clinical benefit (PR and SD) after one cycle but were switched to a different protocol because of gastrointestinal AEs and owner preference. Figure 1 outlines selection criteria and Table 2 details the patient population, which consisted mostly of large-breed, neutered dogs.
Citation: Journal of the American Animal Hospital Association 60, 1; 10.5326/JAAHA-MS-7372
All dogs were initially diagnosed with large cell lymphoma through cytology. Histopathology was performed in 1 dog and confirmed high-grade lymphoma. Immunophenotype was determined via immunocytochemistry in 15 dogs and flow cytometry in 6 dogs. Immunogenotype was determined via polymerase chain reaction for antigen receptor rearrangement Pin 5 dogs. Three dogs had multiple diagnostics performed to determine phenotype; however, immunophenotype was unknown in 5 dogs. The majority (n = 16, 62%) of dogs were not fully staged and determined to be at least stage III based on generalized lymphadenopathy appreciated on physical examination. Abdominal ultrasound was performed on 4 dogs and thoracic radiographs were performed on 5 dogs. Three dogs were determined to be stage IV based on generalized lymphadenopathy and cytologic evidence of hepatic or splenic involvement. Eight dogs (31%) were determined to be stage V based on the presence of lymphocytosis and circulating atypical lymphocytes. The majority of dogs (n = 16, 62%) were substage b at the time of MOC treatment initiation.
Previous Treatment
Twenty-one dogs (81%) were initially treated with the 19 wk CHOP protocol. Two dogs (8%) were treated with single-agent doxorubicin, two dogs (8%) were treated with lomustine, and one dog (4%) was treated with vincristine, cyclophosphamide, and prednisone. Sixteen dogs (62%) achieved a CR, eight dogs (31%) achieved a PR, one dog (4%) achieved SD, and one dog (4%) achieved PD as their best response to initial therapy. The median duration of initial best response was 42 days (range 0–454 days). Cytology confirmed first relapse in 14 dogs (54%), and PD was noted via lymph node measurements in the remaining dogs. The median duration between initial diagnosis and initiation of the MOC protocol was 152 days (range 34–764 days). Two dogs (8%) were treated with MOC as first-line rescue therapy. Twelve (46%) dogs were treated with lomustine as first-line rescue treatment either with (n = 1) or without (n = 11) L-asparaginase. Various other rescue protocols (CHOP-based, single-agent doxorubicin, mitoxantrone, rabacfosadine, L-asparaginase) were used before MOC therapy. Two dogs received L-asparaginase within 1 wk of MOC. The median number of chemotherapy protocols prior to MOC was 2 (range 1–8). Twenty-four dogs (92%) had been treated with vincristine as part of first-line or rescue therapy before MOC initiation.
MOC Protocol
Eleven dogs (42%) were not treated with a steroid concurrently, whereas 8 (31%) were treated with prednisone (0.8–2 mg/kg per os [PO] q 24 hr), and 7 (27%) were treated with dexamethasone (0.10–0.17 mg/kg PO q 24 hr) concurrently depending on clinician preference. All dogs were prophylactically treated with maropitant (1 mg/kg IV) at the time of chemotherapy and for 4 days (2 mg/kg PO q 24 hr to nearest tablet size) following chemotherapy. Sixteen dogs were treated with multiple antiemetics concurrently (n = 13 dogs treated with dolasetron [0.6 mg/kg PO q 24 hr to nearest tablet size] and n = 13 dogs treated with ondansetron [0.5–1 mg/kg PO q 12 hr to nearest tablet size]). Four dogs were treated with an antidiarrhea (metronidazole 10 mg/kg PO q 12 hr to nearest tablet size). The median initial dose of vincristine during the first cycle of MOC was 0.5 mg/m2 (range 0.5–0.6 mg/m2). The dose of vincristine was escalated in 8 of 13 dogs treated with a second cycle of MOC. Three dogs were given cytarabine subcutaneously and the order of treatment was switched in three dogs (melphalan given week 1). The median number of MOC cycles received was 2 (range 0.5–3). Four dogs (15%) completed the planned three cycles. At the time of disease progression and before starting the MOC protocol, nine dogs (35%) were anemic (seven grade 1 and two grade 2) and nine dogs (35%) were thrombocytopenic (five grade 1 and four grade 2).
Toxicity
Twenty-three dogs had adequate follow-up to be included in the toxicity analysis. All 23 dogs experienced AEs, the majority of which were mild and did not require hospitalization. There were no VCOG Common Terminology Criteria for Adverse Events grade 4 or 5 AEs. There was a total of 98 AEs, and 13/23 dogs (56%) experienced both gastrointestinal and hematological AE. AEs are summarized in Table 3. The most common AEs were anemia, neutropenia, and diarrhea. Six dogs experienced multiple instances of anemia, accounting for 70% of anemia AEs. Only one of these dogs was anemic (grade 2) at the start of the protocol. This dog was treated with a blood transfusion during week 2 of treatment and completed a second cycle of MOC before developing PD. The same dog experienced a seizure (VCOG grade 2) 3 days after subcutaneous administration of cytarabine. The dog had no prior history of seizures, and antiepileptic therapy was not initiated. The dog subsequently tolerated an additional dose of cytarabine without experiencing seizures. None of the other dogs required a blood transfusion. Of the seven dogs that were anemic at the time of MOC initiation and had adequate follow-up for toxicity analysis, anemia progressed following MOC treatment in three dogs, remained stable in two dogs, and improved in two dogs.
There were seven dose delays and two dose reductions due to hematologic toxicities. Four dogs experienced multiple instances of neutropenia, accounting for 62% of the neutropenic events. Similarly, four dogs experienced multiple instances of diarrhea, accounting for 47% of diarrhea AEs. In two dogs, MOC was discontinued due to AEs and owner-perceived reduced quality of life. One dog experienced lethargy (grade 1) and diarrhea (grade 3) despite treatment with metronidazole. The other dog experienced anorexia (grade 2) despite treatment with ondansetron and maropitant, leading to discontinuation of the protocol. An appetite stimulant was not prescribed.
Patient Outcomes
The overall response rate for 26 evaluable dogs was 38%, which included 19% CR (n = 5 dogs) and 19% PR (n = 5 dogs). The overall clinical benefit (CR, PR, and SD) was 65%, which included 7 dogs with SD (27%). Two dogs with stage Vb T-cell lymphoma died less than three days after treatment and were assumed to have progressive disease. A physical examination was not performed prior to death, and therefore, these dogs were excluded from toxicity analysis. The other two dogs with T-cell lymphoma met inclusion criteria for response and experienced clinical benefit (CR and SD). The influence of individual factors on response could not be assessed due to low incidence in each subgroup.
The two dogs in which MOC was discontinued due to owner preference were excluded from PFS analysis. The median PFS for evaluable dogs (n = 24) was 29 days (range 1–280 days). The median PFS for dogs (n = 6) experiencing SD was 36 days (range 33–42 days). The median PFS for responders (CR and PR) was significantly longer compared with dogs who did not respond to MOC (47 days versus 14 days; P = .002). Similarly, the median PFS for dogs experiencing clinical benefit (CR, PR, and SD) was significantly longer than that for dogs experiencing PD (37 days versus 14 days; P = .005; Figure 2). Overall survival was not evaluated due to variable pursuit of additional rescue protocols. However, 21 dogs went on to receive additional chemotherapeutics.
Citation: Journal of the American Animal Hospital Association 60, 1; 10.5326/JAAHA-MS-7372
Discussion
The primary objective of this study was to determine the tolerability of the combination of melphalan, vincristine, and cytarabine in a population of dogs with relapsed or refractory multicentric lymphoma. Our results demonstrate that the combination was well tolerated. Hematologic toxicity, specifically anemia and neutropenia, were the most common AEs observed in dogs treated with MOC, and AEs were generally mild.
The frequency of anemia in our study was unexpected. Myelosuppression is a common AE of chemotherapy; however, anemia is a less common AE due to the long lifespan of erythrocytes compared with platelets and neutrophils. Previous studies of protocols using cytarabine have not reported anemia as a side effect. However, the variable use of granulocyte colony-stimulating factor (G-CSF) and/or erythropoietin within several of these studies may have decreased the incidence of hematologic toxicity.26,35 Gillem et al. evaluated dogs with relapsed lymphoma treated with cytarabine and carboplatin without the use of G-CSF and reported anemia in 31% of dogs.10 Ruslander et al. evaluated single-agent cytarabine in treatment-naive dogs with lymphoma without the use of G-CSF.24 Although anemia was not a reported side effect, all dogs’ packed cell volume (PCV) decreased (median 5%, range 2–11%) following treatment. In the population described by Ruslander et al., the median initial PCV was 47% (range 23–57%), whereas in our population, the median initial PCV was 37% (range 24–54%).24 Subsequently, in our study, a mild decrease in PCV was often sufficient to cause a mild anemia. Importantly, the majority of anemia AEs in our study occurred in a subset of dogs (n = 6 of 23) and the risk of significant complications related to anemia was low. The only dog that required a blood transfusion had been anemic at the time of relapse and before initiating MOC (grade 2) and completed an additional cycle of MOC following the transfusion. The frequency of anemia seen in our study may be a consequence of this novel drug combination; however, anemia may also be related to undocumented bone marrow involvement, cumulative bone marrow suppression from previous chemotherapy, or both.
The frequency and severity of neutropenia was reduced in our study compared with previous studies.4,7,8,10,14,15,17,18 Dose delays were uncommon, and the dose of vincristine was escalated in 8 of 13 dogs treated with a second cycle of MOC. The only instance of grade 3 neutropenia was observed in a dog during the second cycle when the dose of vincristine had not been escalated. As the incidence of neutropenia was infrequent and mild, a higher initial dose of vincristine could be considered, which may improve the response rate and duration of response to the MOC protocol.
The third most common AEs were diarrhea and lethargy. Gastrointestinal and constitutional toxicity was relatively uncommon in the MOC protocol compared with other multiagent rescue protocols.6,13–15,17 As this study was retrospective, gastrointestinal toxicity may have been underestimated, particularly if low grade, as it may not have been reported by the owners or noted in the clinical records. Although gastrointestinal AEs were generally mild, the MOC protocol was discontinued in two dogs by owners as a result of an AE or a perceived decrease in quality of life. One of these dogs experienced anorexia, and the other experienced lethargy and diarrhea. These AEs could have been addressed through dose reductions and/or prophylactic medications rather than discontinuation of chemotherapy. Balancing chemotherapy toxicity, tumor response, and patient quality of life remains challenging. A recent retrospective study reported multiagent chemotherapy protocols and hematopoietic tumors are reported risk factors for severe AEs.36 Severe AEs were defined as AEs that cause chemotherapy discontinuation, hospitalization, death, grade 4 hematological AEs, and symptomatic grade 3 hematological AEs. Chavelle et al. reported that severe AEs were seen in 38% of dogs treated with multiagent protocols, which is higher than observed in dogs treated with MOC (8%).36
Although response to treatment was not the primary objective of this study, the overall response rate of 38P% for a median duration of 29 days reported in our study is comparable to other second-line rescue protocols for the treatment of canine relapsed or refractory lymphoma.7,8,17,18 An additional seven dogs experienced SD for a median of 36 days, resulting in clinical benefit in 65% of MOC-treated dogs. In veterinary oncology, where a cure is often unattainable or clinically challenging, clinical benefit should be considered an acceptable measure of treatment outcome. Although a benefit was seen in a majority of patients, the duration of benefit was limited. Our population had a short median duration of response to first-line treatment. Thus, it is possible a longer duration of benefit to MOC may be seen in populations of dogs with more typical responses to induction treatment. Two dogs experienced durable remissions, experiencing a response for >100 days. Both of these dogs had been treated with MOC as first-line rescue. Only four dogs completed the planned three cycles of MOC; the median PFS for these dogs was 81 days (range 42–280 days). It is possible additional cycles of MOC would result in a more durable response for a subset of patients.
In this study, dogs were excluded from toxicity analysis if they did not have adequate follow-up. Three dogs died within 5 days of receiving cytarabine and vincristine. All three dogs had stage Vb lymphoma, had been refractory to CHOP, and were systemically ill before receiving vincristine/cytarabine. These dogs were suspected to have died of lymphoma; however, necropsy was not performed. The exclusion of these dogs may have introduced bias toward a more favorable toxicity profile in dogs treated with MOC.
Limitations of this study include the retrospective nature, small sample size, and heterogeneity of the population. Although all dogs had multicentric lymphoma, full staging was not required at the time of diagnosis or relapse. Additional limitations include variability in drug dosages, order of treatment, and route of drug administration. In this study, a subset of dogs (n = 3) received cytarabine subcutaneously in four doses over 2 days (target total dose 300 mg/m2). This route was chosen depending on clinician and owner preference. Alvarez et al. found no significant difference in response between subcutaneous or continuous rate infusion administration as part of the dexamethasone, melphalan, actinomycin D, and cytosine arabinoside protocol in dogs.3 However, a pharmacokinetic study of cytarabine in healthy dogs showed that subcutaneous administration has a limited ability to maintain steady-state concentrations compared with continuous rate infusions.37 A follow-up study in dogs with meningoencephalitis treated with subcutaneous cytarabine revealed similar plasma concentrations as previously reported.38 Although the plasma concentration of cytarabine necessary to produce a clinical response in dogs is unknown, rapid elimination may result in the drug being less efficacious when administered subcutaneously.37,38 In the present study, it was not possible to compare responses of dogs treated with either route of administration due to the small sample size.
Conclusion
Our findings suggest MOC combination chemotherapy is well tolerated in dogs with relapsed or refractory lymphoma. Additional studies are warranted to validate the potential benefit of the MOC protocol in a larger cohort and explore optimal dosing.
Flow chart provides details of patient screening and inclusion and exclusion from study. AE, adverse event; GI, gastrointestinal; MOC, melphalan, vincristine, and cytarabine; PFS, progression-free survival.
Kaplan-Meier curve depicting effects of clinical benefit and progressive disease on progression-free survival. CB, clinical benefit; PD, progressive disease.
Contributor Notes
