EVALUATION OF A 0.75 X 10 9 /L ABSOLUTE NEUTROPHIL COUNT CUT-OFF FOR ANTIMICROBIAL PROPHYLAXIS IN CANINE CANCER CHEMOTHERAPY PATIENTS.

Running title

Neutrophil cut-off for antimicrobial prophylaxis

KEYWORDS:

Antimicrobial prophylaxis, antimicrobial stewardship, antineoplastic agents, neutropenia, veterinary oncology.

Authors:

Jocelyn Lucy Bisson MA VetMB MRCVS

Quentin Fournier DVM Dip. ECVIM-CA (Oncology) MRCVS

Emily Johnston

Ian Handel BVSc PhD CStat MRCVS

Spela Bavcar DVM Dip. ECVIM-CA (Oncology) MRCVS

Institution

The Royal (Dick) School of Veterinary Studies and the Roslin Insitute, The University of Edinburgh, Roslin, UK, EH25 9RG

Corresponding Author

J. Bisson, The Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Roslin, UK, EH25 9RG

jbisson@exseed.ed.ac.uk

Acknowledgements:

The authors thank the members of the Oncology Service of the R(D)SVS for their assistance in the management of the dogs included in this study.

EVALUATION OF A 0.75 X 10 9 /L ABSOLUTE NEUTROPHIL COUNT CUT-OFF FOR ANTIMICROBIAL PROPHYLAXIS IN CANINE CANCER CHEMOTHERAPY PATIENTS.

KEYWORDS:

Antimicrobial prophylaxis, antimicrobial stewardship, antineoplastic agents, neutropenia, veterinary oncology.

Word Count Main Text: 6082 words

Number of Tables = 6. Number of Figures = 1

ABSTRACT

Absolute neutrophil count (ANC) cut-offs for antimicrobial prophylaxis in veterinary cancer chemotherapy patients are empirical and vary between institutions. Evidence based cut-offs are vital for antimicrobial stewardship, particularly as global antimicrobial resistance rises. The primary objectives of this study were to evaluate the tolerability of a < 0.75 x 109/l ANC cut-off for antimicrobial prophylaxis in dogs after receiving chemotherapy and its impact on antimicrobial prescription. Predicted nadir ANCs (pnANCs) were stratified into 6 groups (< 0.75 x 109/l [receiving antimicrobial prophylaxis], 0.75–0.99x109/l, 1–1.49x109/l, 1.5–1.99x109/l, 2.0–3.59x109/l and 3.6–12x109/l [reference interval]). The incidences of post-nadir febrile neutropenia (FN) and non-haematological toxicity (NHT) were compared between groups. Five hundred and eighty-six pnANCs were recorded for 181 dogs. There were 4 episodes of post-nadir FN and 90 episodes of post-nadir NHT. There was no significant difference in incidence of post-nadir FN (P = 0.063) or post-nadir NHT (P = 0.084) between pnANC groups. Antimicrobial prophylaxis was prescribed following 8.8% of the chemotherapy administrations; had cut-off values of < 1.0x109/l or < 1.5x109/l been used it would have been prescribed in 15.3% and 25.8% of cases respectively. An ANC cut-off of < 0.75x109/l for antimicrobial prophylaxis appears to be well tolerated and minimises the prescription of antimicrobials.

1. INTRODUCTION

Antimicrobial prophylaxis for cancer chemotherapy patients is a controversial subject in human and veterinary oncology. 1,2 Neutropenia is a well-characterised adverse event following chemotherapy and severe neutropenia can predispose patients to infection. 3,4 Antimicrobial prophylaxis is often implemented in asymptomatic neutropenic dogs on documentation of a neutropenia on routine complete blood count (CBC) at the time of the predicted neutrophil nadir. 2,5-7 However, there is limited veterinary evidence as to whether antimicrobial prophylaxis actually benefits neutropenic dogs and no evidence as to what degree of neutropenia requires prophylaxis. 2,8 Consequently, the absolute neutrophil count (ANC) ‘cut-offs’ below which prophylactic antimicrobials are prescribed vary widely between individual clinicians and institutions, with published cut-offs ranging from 0.5x109/l to 2.5x109/l. 2,5-7,9 Most modern veterinary texts recommend antimicrobials for any ANC lower than 1.0x109/l but this remains an empirical value. 3,5,7, In a previous survey conducted at the 2009 Veterinary Cancer Society conference, 4% of veterinarians used an ANC cut-off of < 0.5 x 109/l to prescribe prophylactic antimicrobials, 58% used a cut-off of < 1.0 x 109/l and 29% used a neutrophil cut-off of < 1.5 x 109/l.10 This differs considerably from human guidelines where a cut-off of < 0.5x109/l is routinely recommended in the UK and as low as 0.1x109/l for some patients in the USA. 11,12

As global antimicrobial resistance rises, veterinary antimicrobial stewardship is increasingly important and reducing prophylactic antimicrobials is a key component of this. 13-15 There is also increasing data suggesting that prophylactic antimicrobials may actually be detrimental to some patients, primarily due to effects on the patient’s bacterial microbiome.2

In an attempt to minimize antimicrobial use, the ANC cut-off at our institution for prophylactic antimicrobial administration in asymptomatic dogs, when neutropenia is documented during routine blood work at the time of the predicted post-chemotherapy neutrophil nadir, is < 0.75x109/l. Our group previously published preliminary data from 6 dogs with lymphoma, treated with CHOP chemotherapy protocols suggesting that this cut-off may be safe for clinical use.9 The primary aims of this study were to evaluate the tolerability of this cut-off for prophylactic antimicrobial administration and its impact on antimicrobial usage in a larger cohort of canine chemotherapy patients. Our primary objectives were two-fold: (1) to compare the incidences of post-nadir febrile neutropenia (FN) and (2) post-nadir non-haematological toxicity (NHT) in dogs with ANCs < 0.75 x 109/l receiving prophylactic antimicrobials to those in dogs with ANCs between 0.75 x 109/l and the laboratory reference range (3.6 – 12 x 109/l), not receiving antimicrobial prophylaxis. A secondary objective was to compare the proportion of chemotherapy administrations that would require antimicrobial prophylaxis if various ANC cut-offs were implemented. Our primary hypotheses were that asymptomatic, neutropenic dogs with ANCs between 0.75 – 3.59x109/l, not receiving antimicrobial prophylaxis, would not have significantly different incidences of post – nadir FN or post-nadir NHT compared to dogs with ANCs < 0.75x109/l receiving prophylactic antimicrobials. Our secondary hypothesis was that the proportion of chemotherapy administrations that would require antimicrobial prophylaxis would have at least doubled if a higher cut-off of < 1.5x109/l had been used, compared to the < 0.75 x 109/l cut-off.

2. MATERIALS AND METHODS

2.1. Dogs and Predicted Nadir ANC Selection

The medical records of dogs presented to an academic veterinary specialist oncology service between May 2013 and May 2018, with a cytological or histopathological diagnosis of cancer and treated with conventional dosing regimen chemotherapy, were retrospectively reviewed. Initial staging included complete blood count (CBC) and serum biochemistry, urinalysis, thoracic and abdominal imaging and regional lymph node assessment. Fine needle aspirate of liver/ spleen and bone marrow aspirate were also recommended in dogs diagnosed with haematopoietic tumours but were not always performed.

Predicted nadir absolute neutrophil counts (pnANCs) were obtained from CBCs performed at the time of the most likely ANC nadir of each chemotherapy drug, based on the available literature; 7 days after administration for doxorubicin, epirubicin, lomustine, mitoxantrone, vinblastine and vinorelbine and 10 days after administration for carboplatin (based on standard of care practice at our institution).5

Dogs were excluded if: (1) antimicrobials were prescribed within 24 hours prior to all CBCs for the pnANC; (2) they were clinically unwell at the time of all pnANCs (e.g. VCOG-CTCAE grade 2 or above lethargy, anorexia, diarrhoea, vomiting, pyrexia).

Individual pnANCs were excluded if: (1) the dog received antibiotics within 24 hours prior to CBC (reasons for antimicrobial prescription included bacterial infection, management of chemotherapy-

induced diarrhoea, or inappropriate antimicrobial prophylaxis); (2) the dog inappropriately did not receive antimicrobial prophylaxis; (3) the dog was unwell at the time of CBC; (4) the medical record was incomplete or the documented follow up after the pnANC was less than 1 week.

Only chemotherapy administrations at 2-weekly or 3 – weekly intervals were included. Chemotherapy administrations at weekly intervals (e.g. all vincristine and cyclophosphamide administrations within a 19-week CHOP protocol) were not included as it would be very difficult to distinguish between toxicity related to the predicted nadir of the previous drug and toxicity related to the new chemotherapy administration.

2.2 Chemotherapy protocols

Dogs were treated with a variety of chemotherapy protocols at conventional dosing regimens. Carboplatin was administered as a single agent every 3 weeks (initial dose 300 mg/m2 or 240 mg/m2 if < 10kg).16 Doxorubicin as a single agent every 2-3 weeks or as part of a standardised 19-week CHOP chemotherapy protocol (initial dose 30 mg/m2 or 25mg/m2 if <10 kg).17-19 Epirubicin was administered as a single agent every 3 weeks or instead of doxorubicin in CHOP chemotherapy protocols in dogs with pre-existing cardiac systolic dysfunction diagnosed by echocardiography (initial dose 30mg/m2 or 25 mg/m2 if < 10kg). 20,21 Lomustine was administered as a single agent every 3 weeks (initial dose 70mg/m2). 22 Mitoxantrone was administered as a single agent or as part of a CHOP protocol as a replacement for doxorubicin, in dogs with pre-existing cardiac systolic dysfunction on echocardiography or in dogs that had already received a > 180 mg/m2 total lifetime dose of doxorubicin (initial dose 5.5mg/m2).23,19,24,25 Vinblastine was administered as part of an escalating dose protocol for 8 doses, only those doses in the second half of the protocol (every 2 weeks) were included in the study (initial dose 3.0 mg/m2).26 Vinorelbine was administered as an 8-dose protocol; only the doses in the second half of the protocol (every 2 weeks) were included in the study (initial dose 15mg/m2).27 Ten percent dose reductions were applied if: (1) pre-treatment ANC was < 1.5 x 109/l; (2) pnANC was < 0.75 x 109/l; (3) platelet count was < 50 x 109/l; (4) occasionally due to severe NHT although this reduction was dependent on owner and clinician discretion.ABCB1 - 1δ mutation testing was recommended for predisposed breeds.

2.3 Antimicrobial Prophylaxis and pnANC Stratification

Afebrile neutropenic dogs with pnANC below the cut-off of < 0.75x109/l were prescribed prophylactic trimethoprim–sulphonamide (TMPS) at 15 mg/ kg PO q12h for 3–5 days. The choice of antimicrobials was based on the standard operating procedure in our hospital, established on consideration of a combination of human guidelines and veterinary recommendations. 2,11 The duration of prescription was at the discretion of the clinician and based on the predicted duration of neutropenia for most dogs.5,28 If trimethoprim-sulfonamide was not available or there were concerns regarding adverse effects (dogs with a history of immune-mediated conditions or previous reactions to TMPS) then amoxicillin-clavulanic acid at 12.5 mg/kg PO q12 h or a fluoroquinolone (enrofloxacin at 5mg/kg PO q24h or marbofloxacin at 2mg/kg PO q24h) was prescribed instead for 3 – 5 days. 5,29 Doses of antimicrobials were based on standard manufacturer and formulary recommendations in the UK.30

Predicted nadir ANCs were stratified into 6 groups for comparison and statistical analysis. Group 0 represented the pnANCs below the antimicrobial cut-off, receiving antimicrobial prophylaxis (<0.75x109/l). Groups 1 – 4 represented neutropenias above the antimicrobial cut-off; group 1 (0.75

– 0.99x109/l), group 2 (1.0 – 1.49x109/l), group 3 (1.5 – 1.99x109/l), group 4 (2.0 – 3.59x109/l). Group 5 represented the laboratory reference interval at our institution (3.6 – 12x109/l).

To address the first primary objective groups 0 – 5 were used to compare the incidences of post-nadir FN and NHT. To address the second primary objective group 0, group 0 + group 1 and groups 0 + 1 + 2 were used to assess the theoretical impact of the pnANC cut-offs of < 0.75x109/l, < 1.0x109/l and < 1.5x109/l on the frequency of antimicrobial prophylaxis prescription.

2.4 Post-nadir Toxicity Recording

All dogs were assessed with physical examination, full history and quality of life questionnaire at the time of the predicted neutrophil nadir and again 1 to 2 weeks after the ANC was recorded or sooner if they became clinically unwell. Quality of life questionnaires were filled out by owners and based on the Lynch et al study in 2011 (see supplementary form). 31 Any post – nadir NHT suspected to be related to the chemotherapy administration and occurring after the pnANC, were recorded and graded according to the Veterinary Cooperative Oncology Group criteria for adverse events (VCOG-CTCAE) version 1.1.32 Occasionally the VCOG -CTCAE grading was performed retrospectively although for most cases it had been recorded in the clinical notes at the time of documentation of the toxicity.

Any dog that presented after the pnANC had been recorded with either pyrexia, VCOG -CTCAE grade ≥ 3 NHT or occasionally VCOG-CTCAE grade 2 NHT, had physical examination and repeat CBC performed. However, pnANC and grouping were attributed to the original planned routine CBC and not any subsequent ones. Febrile neutropenia was defined as chemotherapy-induced neutropenia (ANC <2.5x109/l) in conjunction with fever (rectal temperature >39.2°C).28

2.5 Statistical analysis

Chi-squared analysis and Fisher’s exact testing were used to assess the differences in incidence of post-nadir FN and NHT between groups. Equivalence testing and non-inferiority testing were performed to account for the sample size and to assess the clinical significance of the results. 33,34 Predicted nadir ANC groups were sequentially combined for comparison with group 0 to represent each new band of ANC cut-off. The equivalence margin (δ) was set at 10% since a < 10% difference in incidence was considered not clinically significant, as recommended in human guidelines.35,36

Multivariate ordinal logistic regression analysis was used to assess any influence of weight, age, tumour type, chemotherapy agent and chemotherapy dose number on pnANC group. Chemotherapy dose number refers to the chronological number of the dose administered per drug (i.e. 1 = first dose, 2 = second dose etc). Validity of ordinal logistic regression was tested using the method described by Long and Freese (2006)37. Kruskal-Wallis testing and multiple comparisons by the Bonferroni adjustment method were used to test any influence of weight, age, breed, tumour type, chemotherapy agent and dose number on the incidence of post-nadir FN or NHT.

Statistical analyses were performed by commercially available statistics software (Minitab 17, IBM SPSS Statistics 22 and EpiTools epidemiological calculators 38-40), under the guidance of a statistician (IH). A P-value <0.05 was considered statistically significant for all analyses. Charts were made by commercially available software (Microsoft Excel 2016).

1. RESULTS

3.1 Dogs and Distribution of predicted nadir ANCs

Two-hundred and two dogs initially met the inclusion criteria. Twenty-one dogs were excluded leaving a total of 181 for analysis; 7 were excluded due to antimicrobial use at the time of every ANC

(1 dog for recurrent pyoderma, 6 dogs that received metronidazole for diarrhoea prior to every ANC), 4 dogs were unwell at the time of every ANC and 10 dogs were excluded for incomplete medical records. Clinical characteristics of the dogs are presented in Table 1. Of the 15 dogs tested, no dogs were detected to be homozygous or heterozygous for the ABCB1 - 1δ mutation.

Seven hundred and thirteen individual pnANCs were initially selected from the 181 dogs, but 127 ANCs were excluded; 81 due to antimicrobial administration prior to the pnANC or inappropriate antimicrobial administration for ANCs above the cut-off, 30 due to incomplete medical record, 14 as the dogs were unwell at the time of the pnANC and 2 from ANC group 0 due to inappropriate lack of antimicrobial prophylaxis administration. Five hundred and eighty-six pnANCs from 181 dogs were included in the study in total, as presented in Table 2.

Ordinal logistic regression analysis demonstrated that increased weight was significantly associated with higher pnANC groups (odds radio 1.02, 95% confidence interval [CI] 1.01 – 1.04, P <0.0001) but age had no significant influence on pnANC (P = 0.071).

Of the 181 tumours diagnosed 74 were haematopoietic tumours, 26 were histiocytic sarcomas, 25 carcinomas, 15 haemangiosarcomas, 14 mast cell tumours, 14 osteosarcomas, 5 malignant melanomas and 8 miscellaneous tumours. The haematopoietic tumours included 65 multicentric lymphomas, 6 lymphomas of other anatomic form, 2 leukemias and 1 multiple myeloma. The histiocytic sarcomas were localised in 20 cases and disseminated in 6 cases, none were identified as the haemophagocytic variant. The carcinomas included 5 pulmonary carcinomas, 4 intestinal adenocarcinomas, 4 apocrine gland anal sac adenocarcinomas, 3 mammary carcinomas, 3 squamous cell carcinomas, 2 urothelial carcinomas, 2 nasal adenocarcinomas, 1 undifferentiated carcinoma and 1 pancreatic carcinoma. The miscellaneous tumours included 3 soft tissue sarcomas, 2 gastrointestinal stromal tumours, 2 phaeochromocytomas and 1 caecal sarcoma. Most tumours were fully staged according to our recommendations although only 15/71 lymphomas had bone marrow aspirate performed. Ten tumours had evidence of bone marrow involvement including 7 multicentric lymphomas, 2 leukemias and 1 multiple myeloma. The numbers were too small for statistical analysis of the pnANCs in this group. The distribution of the pnANCs per tumour type is presented in Table 3. There was no significant association found between tumour type and pnANC group on ordinal logistic regression (P = 0.935).

The chemotherapeutic agents used in this study are presented in Table 4. The distribution of the pnANCs for each chemotherapeutic agent is shown in Table 5. On ordinal logistic regression analysis treatment with lomustine was significantly associated with lower pnANC groups (odds ratio 0.106, 95% CI 0.041 – 0.278, P < 0.0001) and treatment with carboplatin was significantly associated with higher pnANC groups (odds ratio 3.254, 95% CI 1.023 – 10.370, P = 0.046). A lower chemotherapy dose number was also significantly associated with lower pnANC groups (odds ratio 0.893, 95% CI 0.836 – 0.954, P = 0.001).

3.2 Incidence of post-nadir FN and predicted nadir ANCs.

Overall, 4 episodes of post-nadir FN occurred, representing 0.7% of all chemotherapy administrations and 2% of all dogs included. All dogs had grade 4 neutropenia at the time of diagnosis of FN.

Two of the 4 dogs with FN had group 0 pnANCs recorded and were still receiving antimicrobial prophylaxis with TMPS at the time of development of FN (on day 2 and day 3 after recording the pnANC respectively). One of these dogs had received lomustine prior to recording the pnANC and the other had received doxorubicin. The third dog had a group 2 pnANC and developed FN 2 days

after the pnANC was recorded, this dog had received doxorubicin. The fourth dog had a group 5 pnANC and developed FN 4 days after the pnANC was recorded, this dog had received carboplatin.

There was no statistically significant difference in the incidence of post-nadir FN between ANC groups (P = 0.063, Figure 1) and the incidence was equivalent between all pnANC groups on equivalence testing, indicating that there was no clinically relevant difference between pnANC groups.

There was no statistically significant difference in the incidence of febrile neutropenia between different weights, ages, breeds, tumour types, chemotherapy agents and chemotherapy dose numbers (P = 0.765, 0.847, 0.555, 0.769, 0.502 and 0.253 respectively).

3.3 Assessment of Post-Nadir NHT and predicted nadir ANCs.

There were 90 episodes of post-nadir NHT recorded in 50 dogs. The most common post-nadir NHT were diarrhoea, vomiting, anorexia and lethargy, as presented in Table 6. Other episodes of toxicity were suspected or confirmed infections (11), hepatotoxicity suspected to be related to lomustine (4), cardiotoxicity suspected to be related to doxorubicin (2) and miscellaneous toxicity for which chemotherapy could not definitively be ruled out as the cause (9). Episodes of suspected or confirmed infection were: urinary tract infections (4), perianal pruritus (1 suspected due to fungal infection), pyoderma (1), otitis externa (1), cellulitis (2) and abscess (2). Miscellaneous episodes of toxicity were grade 1 constipation (1), grade 2 pancreatitis (3), grade 1 cough (2), grade 1 tachypnoea (1), grade 1 ataxia (1) and grade 1 tachycardia (1).

There was no significant difference in the incidence of post-nadir NHT between pnANC groups (P=0.084, Figure 1). However, the incidence was not equivalent between pnANC groups on equivalence testing. Non-inferiority testing revealed that groups 1, 1+2, 1+2+3, 1+2+3+4 and 1+2+3+4+5 had a clinically relevant (according to the δ level) lower incidence of post-nadir NHT when compared to group 0.

There was no statistically significant difference in the incidence of post-nadir NHT between different weights, ages, breeds, tumour types, chemotherapy agents and dose numbers (P = 0.368, 0.227, 0.210, 0.429, 0.053 and 0.265 respectively).

3.4 Impact of the < 0.75 x 109/l ANC cut-off on Antimicrobial Prophylaxis Prescription

Using the <0.75 x 109/l ANC cut-off, prophylactic antimicrobials were administered following 8.8% of chemotherapy administrations (52/586) and 18.7% of dogs (34/181) received prophylactic antimicrobials at least once.

Among the 52 presumed nadir ANCs recorded in group 0, TMPS was prescribed in 39 cases, amoxicillin-clavulanic acid in 10, and a fluoroquinolone in 3. Doses and durations of antimicrobial prescription did not significantly deviate from the standard operating procedure of our hospital.

Had the higher ANC cut – off of < 1.0x109/l for prophylaxis been used then antimicrobials would have been administered for 15.3 % of chemotherapy administrations (90/586) and 36.4% of dogs (66/181). Had the cut-off of < 1.5x109/l then antimicrobials would have been administered for 25.8% of chemotherapy administrations (152/586) and 63.5% of dogs (115/181).

3 DISCUSSION

Neutropenic patients or those at risk of chemotherapy-induced neutropenia are frequently

prescribed antimicrobial prophylaxis to try to prevent infections. Studies in humans in the 1960s identified an increased risk of infection when the ANC fell below 2 x 109/l, with patients with ANC < 0.5 x 109/l considered to be high risk and < 0.2 x 109/l very high risk.4 More contemporary human studies define a neutropenia of clinical concern as below 1.0 x 109/l with much lower values typically used as cut-offs for antimicrobial prophylaxis administration. 11,12,41,42

The cut-offs for antimicrobial prophylaxis in veterinary medicine are empirical and vary widely between institutions. 2,6,9 In a previous survey conducted at the 2009 Veterinary Cancer Society conference, 4% of veterinarians used an ANC cut-off of < 0.5 x 109/l to prescribe prophylactic antimicrobials, 58% used an ANC cut-off of < 1.0 x 109/l , 29% used an ANC cut-off of < 1.5 x 109/l and the remaining 9% administered antimicrobials whenever the ANC was below the reference interval.10

Prophylactic antimicrobial prescription in dogs following chemotherapy is controversial. In one double-blinded, placebo-controlled study, dogs receiving prophylactic TMPS at the time of doxorubicin chemotherapy experienced a significant reduction in NHT compared to the placebo group.8 However, another study with epirubicin in dogs found no difference in the incidence of vomiting, diarrhoea, pyrexia or hospitalisation between dogs receiving antimicrobials and those not receiving them.43 There are several reasons to consider limiting the prescription of antimicrobials in chemotherapy patients. First, there is increasing evidence in humans that administering antimicrobial prophylaxis can result in the emergence of resistant bacterial strains, with one literature review documenting resistance to standard prophylactic antimicrobials in 26.8% of pathogens cultured from chemotherapy patients in the USA 44. As global antimicrobial resistance rises both the American Veterinary Medical Association and British Veterinary Association have urged veterinarians to ‘commit to antimicrobial stewardship’ and minimize prophylactic antimicrobial use. 14,15,45 Second, antimicrobials may be associated with adverse effects. Humans receiving antimicrobials with anaerobic activity may be more likely to develop microbial dysbiosis and this has been documented to cause pulmonary complications, inflammatory colitis and Clostridium difficile overgrowth.46 Third, prophylactic antimicrobials may even decrease the efficacy of chemotherapy treatment. Pre-treatment with antimicrobials reduced the efficacy of platinum chemotherapy and cyclophosphamide in some studies in mice although this is yet to be demonstrated in dogs and humans. 47,48

In veterinary medicine institutions typically apply one ‘cover-all’ ANC cut-off to all clinically well dogs when neutropenia is documented at the time of the predicted neutrophil nadir regardless of the age, weight, breed, type of malignancy or chemotherapy protocol etc.2,5,7 This study was deliberately designed to reflect this and the inclusion criteria were broad to ensure that as many dogs as possible for which the cut-off would be implemented in a clinical setting were investigated. There are potential limitations to applying the same ANC cut-off for all dogs, most notably, the fact that in addition to the ANC other factors may increase the risk of FN or other septic complications for individual canine patients following chemotherapy. ANC cut-offs should always be used in conjunction with thorough physical examination of the patient and good clinical judgement.

As hypothesised, there was no statistically significant difference between the pnANC groups for incidence of post-nadir FN or NHT in this study. The absence of a statistically significant difference between the groups for the investigated outcomes could have resulted in a lack of statistical power (i.e. type II error). However, in addition to null-hypothesis significance testing, equivalence and non-inferiority testing were also used. The incidence of FN was equivalent between groups and there was a clinically relevant lower incidence of post-nadir NHT in groups without antimicrobial prophylaxis when compared to those receiving it. This provides robust evidence that a < 0.75 x 109/l ANC cut-off for antimicrobial prophylaxis was well tolerated in this cohort.

In humans one risk factor is protracted duration of neutropenia; the American Society of Clinical Oncology considers patients who are expected to develop an ANC < 0.1 x 109/l for > 7 days as high risk for infection and recommend antimicrobial prophylaxis for these patients.12 Other risk factors in humans include hypotension, respiratory failure, altered mental status, congestive heart failure, arrhythmias, renal failure, age over 60, high disease burden, acute leukaemias, stem cell transplants, patients in the first cycle of chemotherapy and patients on protocols with a high dose intensity. 49 Many human hospitals use the multi-national association for supportive care in cancer (MASSC) index to stratify the patient risk of sepsis and prescribe prophylaxis in a more targeted way. 49

Certain risk factors for the development of FN after chemotherapy have also been identified in dogs including; lower body weight, lymphoma, doxorubicin or vincristine administration or dogs that are homozygous for the ABCB1-1δ mutation.2,7,50 However, there has been little evidence published in veterinary literature so far on how to stratify patient risk.2 The fact that the ANC cut-offs used in veterinary practice do not account for individual risk factors is often used as an argument to be cautious and go for a higher ANC cut-off when prescribing antimicrobial prophylaxis. 5 In this study, increased weight and carboplatin administration were significantly associated with increased pnANC; lomustine treatment and lower chemotherapy dose number were significantly associated with decreased pnANC. The influence of weight on pnANC fits with previous study findings that dogs of lower weight are more at risk of myelosuppression. 50,51 The risk of neutropenia after the first cycle of chemotherapy is well documented in humans and fits with the finding that lower chemotherapy dose number was associated with lower pnANC in this study.49 Neutropenia has been described as the dose limiting toxicity for lomustine in several veterinary studies with ≥ grade 3 neutropenia reported in 71% of dogs treated with an average of 71mg/m2 of FDA approved lomustine in one study.52-54 The median dose of lomustine in this study was 70mg/m2 and it is not surprising that a high proportion (54%) of administrations were associated with ≥ grade 2 neutropenia in this study. Thrombocytopenia and in some cases gastrointestinal toxicity are more commonly reported than neutropenia as the dose-limiting toxicity for carboplatin in humans and in some studies in dogs.55,56 This may explain the higher pnANCs noted for this drug. However, neutropenia has been described as the dose-limiting toxicity for carboplatin in other studies in dogs. 16,57 A delayed neutrophil nadir of ≥ 14 days has been described following carboplatin in dogs and it is possible that in performing a CBC at 10 days for dogs that received carboplatin we missed the true nadir in several cases.55 This theory is supported by the fact that one dog in the study developed FN, with a grade IV neutropenia, 4 days after the recording of a group 5 pnANC and 14 days in total after receiving carboplatin.

We did not find any influence of doxorubicin on pnANC compared to other chemotherapy drugs, despite the fact that it has been previously associated with increased risk of FN in dogs.50 However, there is recent data demonstrating that whilst increased systemic doxorubicin exposure (measured by area under the curve) is significantly associated with degree of neutropenia, it varies widely between individual dogs even with a same 30 mg/m2 dose prescription.58 This variation may have obscured an association with doxorubicin and lower pnANC group in this study. It is also possible that the finding of the previous study was due to confounding as a result of a higher percentage of dogs receiving doxorubicin with lymphoma and/or on combination protocols (high dose intensity) compared to dogs with solid tumours on single agent protocols (lower dose intensity). Whilst 53% of the doxorubicin administrations in this study were for multicentric lymphoma, 54% were from single – agent protocols.

The study that identified doxorubicin administration as a risk factor for FN also identified vincristine administration as a risk factor.50 However, dogs receiving weekly chemotherapy were not assessed in

this study because (1) it would have been difficult to distinguish between toxicity related to the predicted nadir of the previous drug and toxicity related to the new chemotherapy administration; (2) it would have been difficult to account for the variability of chemotherapy administration dependent on the pnANC, since only dogs with an ANC ≥ 1.5 x 109/l would have received further chemotherapy, hampering the comparison with dogs with ANC < 1.5 x 109/l. Therefore, the < 0.75 x 109/l ANC cut-off for antimicrobial prophylaxis was not assessed for cyclophosphamide or vincristine. This could be considered a limitation of this study since these drugs are very commonly used; however toxicity solely related to the pnANC cannot be adequately examined with weekly chemotherapy administrations. Future studies, including two-weekly cyclophosphamide and vincristine administrations, would be needed to investigate if these drugs may require different ANC cut-offs for antimicrobial prophylaxis. There was also a range in dose for some of the chemotherapeutic agents used in the study due to dose reductions for previous haematological or non-haematological toxicity, this may have impacted on the toxicity observed in this study.

There was no significant influence of age or tumour type on pnANC. Haematopoetic tumours have been associated with increased risk of neutropenia in humans and dogs.49,50 This was not the case in this study, possibly due to a difference in the inclusion of the chemotherapy protocols prescribed. In humans, haematopoietic tumours are also often treated with particularly high intensity chemotherapy protocols, including myeloablative regimens. Breeds predisposed to the mutation ABCB1-1δ are at higher risk of developing neutropenias from P-glycoprotein substrate drugs such as doxorubicin.2 Fifteen border collies were included in this study, none of which were detected to carry the mutation. This is not surprising as the frequency of the mutant allele in the border collie population in the UK is reported to be very low.59 We did not have any examples of dogs from other MDR1 mutation risk breeds and therefore could not examine ABCB1 - 1δ as a risk factor for neutropenia in this study. 59

Although weight, chemotherapy dose number, carboplatin and lomustine administration influenced the overall pnANC groups there was no statistically significant difference in the incidence of post-nadir FN or NHT between different weights, ages, breeds, tumour types, chemotherapy agents or chemotherapy dose numbers in this study. This suggests that an ANC cut-off of < 0.75 x 109/l, for clinically well patients undergoing routine blood work after chemotherapy is well tolerated, even when taking into account previously recognised risk factors for FN. This may be because the ANC value is the most accurate factor in predicting the risk of FN compared to other risk factors, especially as the ANC value was not assessed as a confounding factor in previous studies. 50 However, given the low incidence of FN it is possible that a type II error is responsible for the lack of difference found.

It could be argued that the ANC cut-off of 0.75x109/l assessed in this study was too conservative, and could be lowered further to match human guidelines. However, there are reasons to be cautious when considering lower cut-offs. First, clinical signs of FN may be detected much later by pet owners than they would in human patients. Second, pet owners may be more likely to discontinue chemotherapy treatment if side effects occur. Third, the “true” neutrophil nadir may be missed when only one CBC is performed. In the current study, pnANCs were obtained from CBC performed 7 days after administration of the chemotherapy drugs, with the exception of carboplatin which had the CBCs performed after 10 days in line with standard veterinary practice. It is therefore possible that the true neutrophil nadir may have been missed in some dogs. This likely explains why the 2 dogs with pnANCs within group 2 (1.0 – 1.49x109/l) and group 5 (3.6 – 12x109/l) went on to develop grade 4 FN 2 and 4 days later and why carboplatin was associated with higher pnANC groups as discussed above. The possibility of missing the true neutrophil nadir is a limitation of only performing one CBC following chemotherapy. However, there are financial and operational reasons why performing multiple CBCs following chemotherapy is not practical in a clinical setting. Clinicians

may therefore argue that higher ANC cut-offs for prophylactic antimicrobials should be utilized to account for the chance of missing the true nadir.5 However, FN only developed for 2/534 (0.3%) chemotherapy administrations where pnANC ≥ 0.75x109/l. The very low incidences of post-nadir FN and NHT recorded support the protocol of only performing one CBC after chemotherapy and the cut-off of < 0.75 x 109/l appears to be well tolerated.

Additional studies investigating the best strategy to detect the “true” nadir ANC may allow further optimization of antimicrobial prophylaxis and this study should be considered a starting point in finding a suitable ANC cut–off with lower values yet to be investigated. Nevertheless, the reduction in ANC cut-off from < 1.5x109/l (a cut-off used by 29% of veterinary oncologists in one study)10 to the study cut-off of < 0.75x109/l decreased the proportion of chemotherapy administrations requiring antimicrobial prophylaxis by over 50%.

Interestingly, 2 out of 4 of the episodes of FN recorded in this study occurred whilst the dogs were already receiving prophylactic trimethoprim sulphonamide as recommended by the standard operating procedure in the hospital. The development of FN whilst already on antimicrobial prophylaxis has already been reported in the veterinary literature.43,60 In addition, there was a clinically relevant lower incidence of post-nadir NHT in groups not receiving antimicrobial prophylaxis when compared to those receiving it. This could raise the question of the adequacy of the antimicrobial prophylaxis protocol implemented. Since the FN developed at 2 and 3 days after the presumed nadir ANC when the 2 dogs were still receiving prophylactic antibiotics, the duration of the prescription cannot be implicated as the cause and a duration of prophylactic antimicrobials of 3-5 days appears to be appropriate based on this data. It is possible that the TMPS being used for prophylaxis may not have been appropriate. However, in human oncology there is generally an acceptance that one antimicrobial (either TMPS or a fluoroquinolone) is adequate for prophylactic cover and addition of further broad-spectrum cover only leads to increased morbidity and no improvement in mortality.11,61 Both dogs received an average dose of 15 mg/kg q12h TMPS as recommended by the standard operating procedure in the hospital. Doses of antimicrobials were based on standard manufacturer and formulary recommendations in the UK.30 However, it must be recognised that manufacturer and formulary dosing recommendations vary between countries with higher doses typically recommended in the USA for marbofloxacin and amoxicillin-clavulanic acid.62 It is vitally important to prescribe adequate antimicrobial doses both for efficacy and for good antimicrobial stewardship.14 Per the standard operating procedure implemented, the most common antimicrobial used in this study was TMPS (prescribed in 39 cases with ANC < 0.75 x 109/l). Importantly there is no variation in dosing recommendations between the UK and USA for TMPS.30,62

TMPS has been associated with blood dyscrasias, keratoconjunctivitis sicca, hypothyroidism, hyperkalaemia, cholestasis, acute hepatic necrosis and skin disease as well as diarrhoea.29 When a dog was prescribed TMPS, owners were made aware of potential side-effects and clinicians were advised on the potential clinical signs to look out for. There was no TMPS side effect noted in the cohort of dogs included in this study, although no specific testing was actively performed. A prospective study would be needed to compare the efficacy of different antimicrobials for prophylaxis and the doses required in neutropenic canine chemotherapy cancer patients. It is our opinion that the antimicrobial prophylaxis protocol implemented in this study provides a satisfactory coverage whilst presumably decreasing the risk of antibiotic resistance, as only 2/52 dogs (3.8%) developed FN.

This study had limitations. First, because of the retrospective nature of the study, some adverse events may not have been recorded appropriately. We assessed both the outcomes of post-nadir FN and NHT to ensure that other toxicity potentially associated with neutropenia were included in the

analysis. It is possible that not all of the NHT recorded after the pnANC were recorded after the “true” neutrophil nadir. However, any NHT recorded after the pnANC would be atypical for chemotherapy associated NHT as this typically develops in the first 2 – 4 days post chemotherapy administration, and therefore could be indicative of the dog developing infectious complications.

It is difficult with any retrospective study to definitively ascribe the toxicity that occurred to the preceding chemotherapy or ANC. It may have been due to other concurrent conditions, particularly as the cohort included in this study (as is the case with most oncology cohorts) was aged (median age 9.9 years). Additionally, as some of the NHT grading was based on quality of life questionnaires there would have been some inherent subjectivity due to owner perceptions. However, the incidence and grades of toxicity reported in this study is comparable to previous studies.7,9,28,63

Second, the implementation of the ANC cut-off for antimicrobial prophylaxis was not assessed for vincristine and cyclophosphamide chemotherapy administrations, due to an inability to accurately interpret toxicity for these drugs.

Third, despite the 586 chemotherapy administrations included, there was inadequate power to fully analyse all potential risk factors and their influence on the incidence of FN. Further prospective studies assessing the requirement for individual ANC cut-offs according to breed, ABCB1 - 1δ mutation, chemotherapy drug received etc are required.

To our knowledge, this is the first large veterinary study to assess the tolerability of an ANC cut-off for antimicrobial prophylaxis, providing valuable evidence to guide clinicians and implement a more standardized approach to prescription of antimicrobial prophylaxis. The overall incidence of post-nadir FN recorded in the study was only 4/586 (0.7%) of chemotherapy administrations, supporting the good tolerability of the ANC cut-off <0.75 x 109/l in this cohort of dogs. Furthermore, all episodes of post-nadir FN recorded in this study were associated with grade 4 neutropenia (< 0.50x109/l), which supports the hypothesis that the risk of developing FN when ANC ≥ 0.75x109/l is extremely low in canine chemotherapy patients.

CONCLUSIONS

A < 0.75 x 109/l ANC cut-off for antimicrobial prophylaxis in asymptomatic, neutropenic dogs, appeared to be very well tolerated in this cohort of dogs. Only 4 episodes of post-nadir FN were recorded and there was low incidence of NHT recorded after the pnANC. There was no significant difference in the incidence of post-nadir FN or NHT found between pnANC groups. Adopting an ANC cut-off < 0.75x109/l reduced potential antimicrobial prescriptions by more than 50% compared to a < 1.5x109/l cut-off. The data presented in this study supports the adoption of an ANC cut-off < 0.75x109/l for the prescription of prophylactic antibiotics in canine chemotherapy cancer patients. Future studies are needed to investigate the tolerability of lower ANC cut-offs, and the possible benefit of stratifying ANC cut-offs according to individual risk factors for developing FN.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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FIGURE LEGENDS

Figure 1: The incidence of post-nadir NHT and post-nadir FN per pnANC group

Table 1: Characteristics of the 181 dogs included in the study

Table 2: Distribution of 586 predicted nadir ANCs within each ANC group

Abbreviations: ANC, absolute neutrophil count

Table 3: Distribution of 586 predicted nadir ANCs for each tumour type

Abbreviations: pnANC, Predicted nadir absolute neutrophil count

Table 4: Characteristics of the 7 chemotherapeutic agents included in the study

Table 5: Distribution of 586 predicted nadir ANCs for each chemotherapeutic agent

Abbreviations: ANC, absolute neutrophil count

Table 6: Incidence of the most common post-nadir non-haematological toxicities in 181 dogs for each predicted nadir ANC group

Abbreviations: ANC, absolute neutrophil count; VCOG,

All chemotherapy-induced toxicities were graded according to the Veterinary Cooperative Oncology Group criteria for adverse events (VCOG-CTCAE) version 1.1.32 No toxicity ≥ grade 4 were recorded