Objective Cytarabine, a cell-cycle phase-specific antimetabolite, has been reported to improve outcomes in dogs with bone mar- row (BM) or central nervous system (CNS) lymphoma involvement receiving combination chemotherapy. The objective of this study was to evaluate the incidence and severity of toxicity of cytarabine constant rate infusion (CRI) in dogs with high-grade non-Hodgkin lymphoma.
Methods Medical records of canine lymphoma patients with con- firmed or suspected BM (group 1) or CNS (group 2) involvement, treated with a modified cyclophosphamide, epirubicin, vincristine and prednisolone protocol, including a single dose of cytarabine given as CRI, were reviewed and adverse events graded.
Results Twenty-six dogs were included. Gastrointestinal toxicity occurred in 17 dogs (65.3%), with 5 (19.2%) experiencing grade III or IV toxicity. Neutropenia occurred in nine dogs (34.6%), but was grade I or II in most cases. Three dogs (11.5%) had thrombocyto- penia: one grade III and two grade IV. Four dogs (15.3%) experi- enced increases in alanine amino transferase: one each grade I and II and two grade III. Five dogs (19.2%) required hospitalisation to manage toxicity after completing cytarabine CRI, and haematological toxicity resulted in treatment delays in five dogs (median delay of 4 days, range: 3–7 days).
Conclusion Our findings suggest that gastrointestinal toxicity should be expected in lymphoma patients undergoing cytarabine CRI.
Keywords bone marrow; central nervous system; chemotherapy; cytarabine; cytosine arabinoside; lymphoma
Abbreviations ALT, alanine amino transferase; BM, bone marrow; CEOP, cyclophosphamide, epirubicin, vincristine and prednisolone; CHOP, cytoxan, hydroxyrubicin (adriamycin), oncovin (vincristine), prednisone; cNHL, non-Hodgkin lymphoma; CNS, central nervous system; CR, clinical response; CRI, constant rate infusion; G-CSF, granulocyte colony stimulating factor; GI, gastrointestinal; MRI, magnetic resonance imaging; MST, mean survival time; PARR, PCR for antigen receptor gene rearrangement; PD, progressive disease; PR, partial response; RI, reference interval; SD, stable disease
Haematopoietic neoplasias are common in dogs, with canine non-Hodgkin lymphomas (cNHLs) comprising over 80% of all haematopoietic cancers.1,2 Among cNHLs, high-grade B- cell lymphomas prevail and first-line chemotherapy consists of maintenance-free protocols, including prednisolone, vincristine, cyclo- phosphamide and doxorubicin (CHOP); variants replacing doxorubi- cin with epirubicin (cyclophosphamide, epirubicin, vincristine and prednisolone [CEOP]) show similar results.3–6 High-grade T-cell lym- phomas are less common and are associated with lower response rates and shorter remission rates when using CHOP-based protocols.7
Cytarabine (1b-arabinofuranosylcytosine, cytosine arabinoside or ara- C) is a pyrimidine analogue isolated from the sponge Cryptothethya crypta.8 Once introduced into the blood, cytarabine enters tumour cells, where it undergoes phosphorylation through sequential enzy- matic interactions to form arabinosylcytosine triphosphate, the active metabolite. Arabinosylcytosine triphosphate is incorporated into DNA during replication, preventing successful DNA synthesis and interfer- ing with DNA repair. Thus, it is S phase-specific. Cytarabine also inhibits DNA polymerase α and terminates DNA elongation, which ultimately results in cell death.8 Cytarabine and its metabolites are degraded by cytidine/deoxycitidine deaminase to the inactive metabo- lite uracil arabinoside (ara-U) mainly in the liver.8,9
As a result of its short half-life (T1/2 of 1.33 and 1.15 h after subcuta- neous and intravenous administration, respectively) and rapid deam- ination, a steady-state of cytotoxic plasma levels of cytarabine metabolites is best achieved by continuous exposure to the drug.9 Moreover, cellular accumulation and retention of cytarabine also correlates with cytotoxicity and duration of clinical response in human leukaemias.10 This effect can be explained by three hypotheses:
- 1 Most chemotherapeutic agents cause tumour cell death only when cells are actively cycling, and therefore quiescent cells (G0) are not affected. Regardless of the length of the cell cycle, cytarabine given by constant rate infusion (CRI) may increase the chances of killing the cancer cell in the appropriate phase.11
- 2 Because of the short half-life of cytarabine, CRI maintains an effec- tive drug concentration for a longer period of time, improving the therapeutic index.9,12
- 3 Transport into the tumour cell occurs by diffusion and occasion- ally by active transport. This transport may depend not only on the drug concentration but also on the length of time that the drug is exposed to the cell membrane.8,11
Aust Vet J 2020;98:69–78
doi: 10.1111/avj.12895
*Corresponding author.
aDepartment of Small Animal Clinical Science, Institute of Veterinary Science, University of Liverpool, Neston, CH64 7TE, UK; a.guillen@liverpool.ac.uk bSouthfields Veterinary Specialists, Southfields, Laindon Essex, SS15 6TP, UK
Thus, administration of cytarabine as CRI is superior to the subcuta- neous route and both efficacy and toxicity will be affected by the drug dose and duration of administration.9,11–13
In human medicine, cytarabine is important in the therapy of acute myeloid leukaemia, central nervous system (CNS) lymphoma and lymphoblastic leukaemia.14–16 A phase II trial in humans with CNS lymphoma showed that the addition of cytarabine to a methotrexate single agent protocol significantly increased response rate and overall survival.15
In domestic animals, the main uses are treatment of lymphoproliferative diseases and meningoencephalitis of unknown aetiology.17–24 Despite poor efficacy as a single agent in canine lymphoma,23 cytarabine may have a role to play in CNS (stage V) lymphoma, as it crosses the blood brain barrier.12,25,26 In addition, cytarabine intensification of induction protocols was associated with increased survival in a small number of patients with bone marrow (BM) infiltration.22
In humans, treatment duration and drug concentration influence the incidence of toxicity. The most common toxicities are myelo- suppression, oral mucositis, diarrhoea, ileus, idiosyncratic reactions leading to fever, and elevation of liver enzymes.13,14 In dogs, the most commonly reported adverse events are BM and gastrointestinal (GI) toxicity,22,23 although the limited data often relates to subcutane- ous administration and/or use in multi-agent rescue protocols. Calci- nosis cutis and infiltrative lung disease have also been described.27,28
To date, only two studies have specifically reported toxicity data after CRI cytarabine. Ruslander et al. evaluated single agent cytarabine administered as a CRI, as first-line treatment in 15 dogs with lymphoma. A total dose of 600 mg/m2 was given over 48 h. No objective responses were observed but toxicity (graded subjectively) included mild to moderate GI toxicity in six of 15 dogs, severe diarrhoea in two dogs, epistaxis in one dog, lethargy in two dogs and haematological toxicity (mild in four dogs, moderate in five and severe in one dog), though no hospitalisation was required.23 Sub- sequently, Marconato et al. evaluated the addition of cytarabine to a maintenance-free vincristine, cyclophosphamide, asparaginase and doxo- rubicin based protocol in dogs with naive lymphoma and BM involve- ment. For nine dogs treated with cytarabine CRI at 150 mg/m2 per day over 24 h for five consecutive days, the median survival time was signifi- cantly longer compared with eight dogs that did not receive cytarabine (243 versus 72.5 days). Reported toxicity was limited to two grade II GI events and one grade I haematological toxicity.22 These contradictory results may be in part due to the differences in dose and duration of treat- ment, and differences in data recording in these retrospective studies. The use of granulocyte colony stimulating factor (G-CSF) in Marconato et al.’s study may have decreased the incidence of haematological toxicity, but does not explain the low frequency of significant GI toxicity.
Given these conflicting data, the purpose of this retrospective study was to describe the toxicity profile of CRI cytarabine given as part of the first-line treatment for high-grade cNHL with confirmed or suspected BM or CNS involvement. Based on clinical experience and preliminary clinical audit of a small number of cases, our hypothesis was that cytarabine CRI caused a higher incidence of GI toxicity compared with previous reports. A secondary objective was to assess clinical responses and to look for prognostic factors in our groups of dogs. Survival data are also reported.
Material and methods
Study population
For this retrospective case series, the computerised clinical records database of the hospital was searched for patients that had received cytarabine as a CRI between 2012–2017. Dogs were included if: (1) they had a cytological or histopathological diagnosis of high- grade cNHL, (2) cytarabine CRI was included as part of the first-line CEOP chemotherapy protocol5 to treat naive dogs and (3) pre- and post-treatment haematology results were available.
Dogs were excluded if they had received cytarabine subcutaneously or had incomplete records.
Dogs in group 1 had confirmed or suspected BM lymphoma, based on flow cytometry or cytology of BM aspirates: samples were reviewed by a board-certified clinical pathologist and a positive result was reported when >3% of nucleated cells were classified as neoplas- tic lymphocytes, as previously established.29 As Graff et al. demon- strated a correlation between the presence of neoplastic lymphocytes in the blood smear and the presence of BM involvement in dogs with lymphoma, some patients were presumptively diagnosed with BM infiltration when haematology showed lymphocytosis (>3.8 × 109/L, i.e. greater than the upper value of our internal laboratory reference interval [RI]) with a predominance of atypical lymphoblasts on blood smear examination.30 Neoplastic cells were evaluated for CD34 expression, to confirm lymphoma (CD34 negative) rather than acute lymphoblastic leukaemia, when appropriate.31
Dogs in group 2 had confirmed or suspected CNS lymphoma. The diagnosis of solitary CNS lymphoma was based on CSF analysis, confirming the presence of neoplastic lymphocytosis or a monomor- phic population of blast cells, in combination with magnetic reso- nance imaging (MRI) findings. PCR for antigen rearrangement (PARR) was performed on CSF samples to assess for clonality if cytology was equivocal. Dogs were presumptively diagnosed with CNS lymphoma as part of multicentric disease when they presented with multicentric lympho-proliferative disease and neurological signs deemed likely to be due to lymphoma.32
Information regarding signalment, tumour location, disease stage and substage, previous drugs given, adverse events, drug protocol and survival data were obtained from the records and / or by tele- phone follow-up. Staging work-up for all dogs included physical examination, total blood cell count, biochemistry, thoracic and abdominal imaging (either by computed tomography, radiography or ultrasonography) with or without fine-needle aspirates of abnor- mal lymph nodes, liver and spleen. All dogs were staged according to the World Health Organisation clinical staging system.33 In both groups, immunophenotyping was performed by flow cytometry (on blood, lymph node or BM samples) or immunohistochemistry to distinguish between B-cell and T-cell immunophenotypes, at the cli- nician’s discretion.
Cytarabine protocol and administration
All dogs received cytarabine (Cytarabine®, 100 mg/mL, Hospira Limited, Maidenhead, UK) as part of a modified 25-week discontin- uous CEOP protocol due to suspected or confirmed BM (group 1) or CNS (group 2) lymphoma involvement. CEOP is the standard
protocol for lymphoma in our institution.5 Cytarabine was given once during the induction phase in the first, second, third and fourth week of the protocol in an attempt to intensify the treatment in both groups.
Cytarabine was diluted in saline (0.9% NaCl) and administered via a burette giving set and infusion pump over 8, 12 or 24 h, for one to four consecutive days. Total dose ranged from 100 to 450 mg/m2. Treatment over the study period was not standardised. G-CSF (filgastrim, Neupogen® 60 MU/0.5 mL; Dompe Biotec, Milan, Italy) was given prophylactically before or during the treatment course, at the clinician’s discretion.
Toxicity evaluation
All patients were evaluated 7–10 days after the first day of the infusion of cytarabine by history, physical exam and haematology. Biochemis- try was analysed at the discretion of the clinician. Adverse events were determined from the medical records, owner reports (during the con- sultation or via telephone) and clinical notes and were evaluated according to the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTAE, 2011).34Toxicity was further classified as immediate (during the hospitalisation period for cytarabine treatment) or delayed (after discharge).
Response evaluation
As true response evaluation was impossible based on the available data, crude response evaluation criteria were developed, based on existing VCOG criteria and previous studies.26,35 Complete clinical response (CR) was defined as disappearance of all measurable lesions, normalisation of lymphocyte count with no detectable lym- phoid blast cells on peripheral smear examination and/or complete resolution of neurological signs. Partial response (PR) was defined as ≥30% reduction in the size of measurable lesions, reduction in lym- phocyte count to within the RI with the persistence of <5% imma- ture cells in peripheral blood or any improvement in neurological function without return to normal. Stable disease (SD) was defined as <30% reduction or <20% increase in the size of measurable lesions, the persistence of >5% immature cells in peripheral blood with <20% increase in abnormal cell count, or no change significant change in neurological status for at least 14 days. Progressive disease (PD) was defined as ≥20% increase in the size of measurable lesions or development of new lesions and increase of >20% in circulating neoplastic cells, or deterioration of neurological signs. Response assessment was based on evaluation pre-cytarabine and 7–10 days after treatment. Due to inconsistent restaging, progression free inter- val could not be accurately documented.
Statistical analysis
Overall survival time was calculated from the day of diagnosis to death or euthanasia or last follow-up. Descriptive statistics consisted of median values for continuous data and frequencies for categorical data. Continuous variables were analysed for prognostic factors with a Cox regression analysis. Kaplan–Meier survival curves were per- formed to describe the outcomes between group 1 (BM) and 2 (CNS) using the SPSS 13 software (SPSS 13.0, SPSS Inc, IBM, Chi- cago, IL, USA). For all tests, a P value <0.05 was considered statisti- cally significant.
Results
© 2019 Australian Veterinary Association
Australian Veterinary Journal Volume 98 No 3, March 2020 71
Study population
Between 2012 and 2017, 31 dogs with lymphoma were treated with cytarabine CRI: 26 dogs met the inclusion criteria. Five dogs were excluded: two died of suspected tumour lysis syndrome during CRI, one was euthanased due to disease progression prior to re-evaluation and in two dogs the cytarabine dose was not recorded. Breed and age data for the 26 dogs included are summarised in Tables 1 and 2. The median age at diagnosis was 7 years (range: 1–12 years) with a median weight of 23.7 kg (range: 6.2–54.6 kg).
Staging was performed by computed tomography scan of the thorax and abdomen in seven cases, 17 dogs had thoracic radiographs, 14 had abdominal ultrasound and 7 had abdominal radiographs. MRI of CNS and CSF analysis was performed in four dogs (Table 2). Fine-needle aspirates of the liver or/and spleen were performed in 18 dogs and were consistent with lymphoma infiltration in both organs in 13 dogs, in the liver only in one dog and in the spleen only in another dog. BM aspiration was performed in 12 dogs (Table 1).
Only two dogs were considered asymptomatic (substage a) at presentation: the remainder were substage b. Immunophenotype was available for 22 of 26 dogs: 18 dogs had B-cell lymphoma, three dogs T-cell lymphoma and in one dog the result was inconclusive (nega- tive for CD3, CD8, CD4 and CD21). One dog with B-cell phenotype was negative for CD3, but was (presumed aberrantly) positive for CD8 on flow cytometry.
Group 1: BM involvement
Eighteen dogs were included in group 1: Twelve dogs had confirmed BM lymphoma and six had suspected BM infiltration. The most common clinical signs were lymphadenopathy (13), lethargy (7), weight loss (5), hyporexia (6), diarrhoea (4), cough (2), vomiting (3) and pyrexia (2). In ten dogs (55.5%), haematology revealed lym- phocytosis of greater than 3.8 × 109/L (median lymphoid cell count of 43.5 × 109/L, range: 7.59–104) with a predominance of abnormal blasts in circulation. Five of 10 dogs with lymphocytosis had con- firmed BM lymphoma and in the other five dogs this was suspected. Additional diagnostic tests in the six dogs with suspected BM infil- tration included: lymph node flow cytometry in five dogs, one of which also had flow cytometry on blood, and another which also had lymph node immunohistochemistry. The remaining dog had lymph node immunohistochemistry. Dog 9 was suspected to have severe BM involvement based on pancytopenia. Acute lymphoblastic leukaemia was considered highly unlikely in all these dogs based on flow cytometry (absence of CD34 expression), presentation and dis- ease progression (Table 1).
Group 2: Dogs with CNS involvement
Eight dogs were included in group 2: Four dogs had solitary CNS lymphoma diagnosed by CSF analysis and MRI findings, and addi- tional PARR testing in two dogs. These four cases had no lymphade- nopathy or other organ involvement. Four dogs had CNS involvement as part of multicentric disease, based on the clinical pre- sentation (concurrent lymphadenopathy) and improvement of the neurological signs after chemotherapy treatment. None of the dogs
had a history of travel outside the UK. Based on the neurological exam, the lesion was localised in the forebrain in two cases, multifocal (brain and spine) in one case, thoraco-lumbar spine in two dogs, ocular nerve in two dogs and other cranial nerves in one dog. Clinical signs and results of MRI scans are summarised in Table 2.
Cytarabine treatment
Cytarabine CRI was delivered over 8 to 72 h during hospitalisation. Total dose ranged from 100–450 mg/m2 (median: 300 mg/m2). Median duration of hospitalisation, including the time for treatment delivery and additional time for management of immediate side effects, was 3 days (range: 1–11 days).
Cytarabine was given as the first treatment (week 1 of the protocol) in 16 dogs, in the second week of the protocol in four dogs, in the third week in three dogs and in the fourth week in three dogs. Fourteen dogs received L-asparaginase concurrently with cytarabine. Previous drugs received included vincristine, cyclophosphamide, L-asparaginase and in one dog, rabacfosadine. Due to anticipated neu- tropenia, 13 dogs (50%) were also treated prophylactically with 5 μg/ kg of G-CSF subcutaneously for three consecutive days (Table 3).
Overall toxicity
A total of 65 toxicity events were recorded (Table 4) for 17 patients. These included mainly GI effects (24 events) and myelosuppression (12 events). Only nine dogs did not experience any adverse effects.
No infusion-related reactions or anaphylactic events were observed during infusion. One dog had erythema and two had phlebitis at the
infusion catheter placement site after treatment, which caused a short episode of lameness. Two patients developed fever (>39.3 C) during the treatment which was not associated with neutropenia; this could be drug-related or a result of a systemic inflammatory response to tumour lysis.
Neutropenia was recorded in nine patients (34.6%): five dogs had grade I, three dogs grade II, and one dog grade IV. Seven of these nine dogs received cytarabine as the first drug of the protocol, but the other two had received vincristine within the previous 7 days. No dog developed sepsis.
Thrombocytopenia was observed in three dogs (11.5%): one dog had grade III and two dogs grade IV. One dog had grade IV neutropenia and grade III thrombocytopenia but was pancytopenic at diagnosis (pre-treatment neutrophil count 0.45 × 109/L RI: 3–12, and platelet count 65 × 109/L RI: 150–400) due to suspected severe BM involve- ment. Another dog that developed grade IV thrombocytopenia had also a decreased platelet count (57 × 109/L) before receiving treat- ment. None of these events were associated with clinical bleeding.
Sixteen dogs (61.5%) experienced anorexia, vomiting, diarrhoea, coli- tis or melaena. In two cases, due to persistent melaena, the patients developed severe anaemia, that was not associated with thrombocy- topenia or coagulation abnormalities, which led to a packed red blood cell transfusion.
Nine dogs (34.6%) had immediate toxicity and developed diarrhoea while hospitalised. One dog continued to have intermittent diarrhoea at home for a week despite symptomatic treatment with metronida- zole, and seven (26.9%) developed delayed GI signs after discharge.
GI toxicity was graded as moderate or severe (grade III and IV) in five dogs (19.2%) with three of them having received concurrent L- asparaginase (dogs 1, 2 and 16). Diarrhoea developed in 8 of 14 dogs (57.1%) that received concurrent L-asparaginase, and in five out of 12 dogs (41.6%) that did not.
In 12 of the 16 dogs with GI toxicity, side effects were managed symptomatically on an outpatient basis with gastroprotectants, anti- emetics and appetite stimulants. Antibiotics were dispensed according to clinician preference due to concerns regarding concur- rent neutropenia or GI barrier disruption. Five dogs were hos- pitalised: the initial hospitalisation period was prolonged due to toxicity at the end of infusion in four dogs, and two of these dogs were readmitted due to profuse and continuous diarrhoea after dis- charge from the hospital. The fifth dog was discharged uneventfully but then readmitted with grade III diarrhoea.
Increases in alanine amino transferase (ALT) occurred in four of five dogs in which it was measured. Lymphoma infiltration was con- firmed cytologically in one dog, in which a grade III ALT elevation was seen. In another dog with grade III hepatotoxicity, ultrasonogra- phy and liver aspirates revealed neither vacuolar hepatopathy nor lymphoma, so this was attributed to cytarabine toxicity. Grade I and
II elevations were seen in two further dogs but no further investiga- tions were performed.
Urinary complications after cytarabine included two patients with urinary tract infections and one dog developed self-limiting glycos- uria which, in the face of normoglycaemia, was attributed to acute renal tubular damage.
One patient was reported to be tachypnoeic and exercise intolerant after the treatment; however, thoracic radiographs revealed no car- diac or pulmonary abnormalities and the clinical signs resolved within days.
Haematological toxicity resulted in treatment delays in five dogs with a median delay of 4 days (range: 3–7 days): no dose reductions were performed for the next drug given.
Responses and survival analysis
Overall, 21 of the 26 dogs had peripheral lymphadenopathy: based on lymph node assessment after cytarabine CRI, 4 dogs achieved CR, 11 PR and 6 SD. When analysing the response based on the lymphocyte count, 13 dogs had lymphocytosis and three more dogs had a normal lymphocyte count with circulating neoplastic
lymphoid cells on smear evaluation. After cytarabine CRI there were
five CR, three PR, five SD and three PD.
In group 1, 17 of 18 dogs had peripheral lymphadenopathy and there were three CR, nine PR and five SD. Two dogs that had CR had received vincristine 7 days prior to the CRI and were already in PR. When analysing the response in the 12 dogs with lymphocytosis or abnormal lymphoblast in circulation in that group, there were five CR, one PR, four SD and two PD. Dog 8 had a worsening of the lymphocy- tosis (PD) despite having a PR in the peripheral lymph nodes. On the other hand, dog 14 had a CR based on lymphocyte count with SD in the peripheral lymph nodes. Responses are summarised in Table 5.
In group 2, four out of nine dogs presented with concurrent lymphade- nopathy and there was one CR, two PR and one SD. Four dogs had lymphocytosis or abnormal circulating lymphocytes and there were one CR, two PR and one SD. Six of nine dogs (66.6%) experienced partial or complete responses based on neurological examination (Table 3).
Only one dog was still alive at the time of writing and was censored from the survival analysis. Overall median survival time was
101 days (range: 21–573 days). Median survival time for dogs in group 1 was 107 days (range: 21–573 days) and in group 2 was 76 days (range: 36–336 days) (Figure 1). After excluding dogs with a presumptive diagnosis in both groups, survival was similar: 113 days (range: 21–573 days) for 12 dogs in group 1 and 48 days (range: 36–336 days) for four dogs in group 2.
In group 1, the median survival time for dogs with lymphocytosis (87.5 days) was shorter than for dogs with a normal lymphocyte count at diagnosis (154 days). However, neither the presence of lym- phocytosis, sex, body condition score, age, development of neutrope- nia, development of GI toxicity nor the total cytarabine dose or dose intensity was statistically associated with survival in our populations of dogs.
Discussion
Cytarabine is a potent inhibitor of DNA synthesis and repair in rap- idly dividing cells, and prolonged exposure of the intestinal barrier to this drug is expected to cause enterocyte apoptosis and villous
atrophy leading to a loss of integrity of the intestinal mucosal barrier, increased permeability and a higher likelihood of bacterial transloca- tion from the GI tract.8,13 This is consistent with the high incidence of GI toxicity in the present study, with 16 dogs (61.5%) experiencing anorexia, vomiting, diarrhoea, colitis and/or melaena. Outpatient symptomatic treatment was given in 12 dogs (46.1%) but five dogs (19.2%) required hospitalisation. Although L-asparaginase has also been associated with GI adverse events as a single agent,36 no differ- ence was found in toxicity incidence or severity between dogs that had received L-asparaginase concurrently or not. Similarly, all dogs were receiving prednisolone at the time of cytarabine CRI, and the contribution of this medication to the overall GI toxicity is uncertain.
Interestingly, the incidence of GI adverse events was similar to the 53.3% reported by Ruslander et al. Conversely, in Marconato’s et al.’s study, toxicity was limited to grade II inappetence and vomiting in two out of nine dogs, but no diarrhoea was reported. Neither of these two studies used prednisolone and the VCOG criteria was not available when those were performed, but neverthe- less the current study is more consistent with Ruslander et al.’s findings.
Neutropenia occurred in nine patients (34.6%) and this led to treatment delays in three dogs (two dogs due to grade II and one grade III toxic- ity). In seven of these nine, neutropenia occurred despite receiving G-CSF concurrently with cytarabine. This incidence of neutropenia is lower than in Ruslander et al.’s study (66.6%), where no stimulating fac- tor was used, but higher than in Marconato et al.’s study (11.1%) where G-CSF was regularly prescribed. This may be because in the Marconato et al.’s study, G-CSF was always given the first day of treatment, while the dogs in our study received G-CSF 24–48 h after starting cytarabine CRI. Interestingly, G-CSF has been reported to cause diarrhoea in humans, but this is not recognised in dogs.37 G-CSF was used in fewer patients in the current study compared with Marconato et al.’s study, so it seems unlikely that this contributed to the more frequent GI toxicity.
Thrombocytopenia occurred in three dogs (11.5%) but in two, pre- treatment thrombocytopenia was present and therefore this could not be exclusively attributed to cytarabine. In the other dog, no clini- cal consequences or treatment delays were observed.
Increases in ALT occurred in four of five dogs in which it was mea- sured, but in three dogs it was not clear if this was attributable to
cytarabine toxicity, as lymphoma infiltration or other hepatopathies could have also contributed to the hepatocellular damage. Hepato- toxicity has been described in experimental studies with rodents and as idiosyncratic reactions in humans, as the liver is the main organ of metabolism, but further work is required to assess this in dogs.9
Neurological toxicity is also a well-documented complication of cytarabine therapy in humans, most commonly seen in rapid infu- sions of high doses of cytarabine (1–2 g/m2) or with high life time cumulative doses.10,14 This can include seizures, cerebellar toxicity, peripheral neuropathies and generalised encephalopathy.13 Although this was difficult to assess in our cohort of dogs with CNS lym- phoma, no records of neurological signs were found in the popula- tion of dogs with BM involvement, which might be explained by the lower doses used in veterinary patients.
Reported responses rates for cytarabine CRI are contradictory, with Ruslander et al.’s study reporting no clinical response to single agent therapy, and Marconato et al.’s study observing CR in eight out of nine dogs (88.8%). Inconsistency between those two studies could be
attributed to the small sample size of the groups or a shorter treat- ment duration in the former (2 days versus 5 days). Although response assessment was not a primary objective of the present study, CR and PR in peripheral lymph nodes were observed in 19% and 52.4% of the dogs, respectively. Moreover, when assessing the response based on the lymphocytosis and/or presence of neoplastic lymphocytes in circulation, 31.2% of the dogs had CR and 18.7% had a PR after cytarabine.
Despite the responses seen, the outcome and mean survival times for group 1 (MST: 107 days) remain disappointing compared with those reported by Marconato et al.’s (243days when treated with cytarabine versus 72.5 days without). Caution must be exercised interpreting the data, as both studies report small cohorts and study design differs. The current study used a modified CEOP protocol, compared with a modified CHOP protocol in Marconato et al.’s study: previous work has suggested equivalent survival times.5 Poorer survival in our population could reflect a shorter infusion duration and lack of standardisation of the protocol in our group of patients.
Figure 1. Kaplan–Meier curve of survival for dogs in group 1 (bone mar- row lymphoma) and group 2 (central nervous system lymphoma) treated with cytarabine constant rate infusion and combination chemotherapy.
Additionally, the use of steroids in our protocol may have contrib- uted to an increased expression of multidrug resistance transporters early in the disease.4
From first principles, the addition of cytarabine is considered benefi- cial in the treatment of canine CNS lymphoma, due to its ability to cross the blood brain barrier when the majority of other drugs do not.8 Moreover, cytarabine remains a mainstay of treatment of CNS lymphoma in humans.16 Recently, LaRue et al. reported outcomes in 18 dogs with primary CNS lymphoma treated with a variety of dif- ferent modalities, including four dogs receiving cytarabine, two of which received the drug intrathecally.25 As only 15% of cytosine ara- binoside is found to concentrate in the CSF compared with plasma concentrations in healthy dogs,12 intrathecal administration may be a way to enhance efficacy while improving the toxicity profile.38 The apparently longer MST of 171 days (range: 1–1942 days) reported by LaRue et al., compared with the MST for group 2 in our study of 76 days (range: 36–1019), could be partly explained by the two dogs treated intrathecally achieving survivals of 113 and 268 days.
While there is evidence that cytarabine improves outcome for CNS lymphoma,25,26 this is not clearly the case for BM involvement.22 In dogs with BM lymphoma, especially those with a large tumour bur- den, one concern is the loss of treatment intensity during the induc- tion protocol due to the substitution of cytarabine for other agents, delaying progression through the standard protocol. This is poten- tially exacerbated by treatment delays in patients with toxicity. It may be that cytarabine is better given later on in the protocol, after achieving complete remission, where it has been shown to play an important role in the consolidation therapy and preventing early relapses in myeloid and lymphoid neoplasms in humans.10,16 Further randomised, prospective studies are needed to further clarify if the addition of cytarabine CRI provides a survival benefit or not.
This study has several limitations, mostly derived from its retrospec- tive nature. Staging was incomplete in some cases and diagnosis of BM or CNS involvement was presumptive rather than confirmed. However, as the primary aim of the study was to assess toxicity, this is less relevant. Although Graff et al. demonstrated a correlation between the presence of neoplastic lymphocytes in peripheral circu- lation and the presence of BM involvement in dogs with lymphoma,30 lymphoma overspill is another explanation. Although overspill is an uncommon and poorly investigated phenomenon in veterinary medicine, it is anecdotally reported mainly in patients with large tumour burdens, as in humans with end stage lympho- mas.2 In addition, CD34 expression was not consistently assessed, and although based on the results of cytology, flow cytometry, clini- cal presentation and response, stage V lymphoma was the most likely diagnosis in five patients, this could not be definitively con- firmed.31,39 Pragmatically, this would impact on survival time but less on toxicity, particularly GI toxicity.
In CNS lymphoma, antemortem diagnosis is usually based on the combination of CSF analysis, MRI findings and PARR. Histopatho- logical confirmation, performed post-mortem in the majority of cases, is still considered the gold standard.25,32 Although clinical signs, investigations and response to treatment supported lymphoma diagnosis in our cohort of dogs, not all of them were tested for infec- tious agents, as these are not considered likely in dogs that had never travelled outside the UK.
An important limitation of the current study is that cytarabine treat- ments were not standardised. However, the protocols used involved lower doses and shorter duration of infusions than reported by Mar- conato et al. and this should have resulted in lesser overall toxicity than previously reported, but instead, adverse effects were common. Conversely, relying on record review may underestimate adverse events if the clinician considered the symptoms not to be clinically significant or if the owner did not report them.
In conclusion, this study showed that GI toxicity is common and should be anticipated in dogs treated with CRI of cytarabine. The addition of this drug in the multidrug CEOP has been recommended in dogs with BM and CNS lymphoma but further larger prospective studies are needed to better assess the potential benefits.
Conflicts of interest and sources of funding
The authors declare no conflicts of interest or sources of funding for
the work presented here.
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(Accepted for publication 27 October 2019)
