Outcomes of different protocols

For canine multicentric lymphoma, chemotherapy remains a primary treatment which has been by far the most effective way to achieve the best response and prolong the survival time to the most in these patients. Reported response rate generally ranges from 89% to 100% [1, 27, 30, 49, 52, 53]. Different protocols had been proposed, and the efficacy of a protocol sometimes could be only assessed by comparison with that from historical or published data due to lack of a control group in the same study. To the author’s knowledge, this should be the first study focusing on the comparison of the two widely used protocols for canine multicentric lymphoma, namely the modified 25-week UW-Madison protocol and the 15-week maintenance-free CHOP protocol, in the same study. In the present study, 42 dogs were given the 25-week protocol and 22 dogs were administered the 15-week protocol. The objective response rate of 15-week group (100%) was not significantly different from that of 25-week group (98.4%), and both were comparable to previous data of response rate in chemotherapy reported for canine multicentric lymphoma. This finding may more likely reflect and be related to the chemosensitive nature of lymphoma as long as chemotherapeutic agent, especially a multiagent protocol, is administered. In the research by Hosoya et al. [52], the substitution of doxorubicin with cytosine arabinoside in protocol did result in a significantly shorter median duration of first remission (94 days versus 174 days, P

<0.01), but such change actually didn’t influence the initial response (92% versus 100%). Modification of protocol in a multi-agent chemotherapy setting for canine

One of the primary intentions of this study is to see whether a more dose-intense and shorter protocol contributes to a comparable or even better outcome, through the setting of two-armed comparison. The 15-week protocol used in this study was first investigated in the study by Curran and Thamm [1]. In that study, the objective response rate was 98%, the median progression-free survival (PFS) was 176 days and the median OST was 311 days. A relatively short PFS was noted. Possible explanation provided by the authors was the fact that 50% of the patient population was substage b, hypercalcemia or stage V at the time of diagnosis, possibly indicating a population with poorer prognosis. Interestingly, there was also 50% of population in the present study met at least one of the criteria mentioned above possibly associated with a poor prognosis. However, the TTP and OST from the 15-week group (217 days and 326 days, respectively) here were longer than the data by Curran and Thamm above, and both were comparable to those from our 25-week group. Even so, it should be interpreted with caution when the comparison was made between studies which may be possibly highly biased due to different backgrounds and numbers of populations, difference in standard of diagnostic, staging and treatment procedures between institutions and the methods of response assessment. Overall, the outcome of a more dose-intense and shorter protocol was acceptable and comparable according to the data presented here.

The median TTP was 217 days for the 15-week group and 242 days for the 25-week group (P=0.503), without significance noted. However, among the censored individuals, dogs that hadn’t reached progressive disease by the closure of this study were more frequently presented in the 15-week group with significance (4.8% in the 25-week group versus 22.7% in the 15-week group, P=0.029), although the distribution of all the censored data for TTP among two groups was not significantly different (P=0.08). This finding indicates that there is still possibility that the median TTP from

the 15-week group potentially may be longer than the data currently available.

Nevertheless, if that’s the case, whether significance in TTP between two groups will exist is still not known. The same situation was noted in the analysis of median OST as well. For OST, all the censored data and those who were still alive and thus censored were both significantly more presented in the 15-week group (for the former 23.8% in the 25-week group versus 50% in the 15-week group, P=0.034; for the latter 7.9% in the 25-week group versus 40.9% in the 15-week group, P=0.002). Although the numbers of the median OST in both group (354 days for the 25-week group and 326 days for the 15-week group) were comparable to the previous published data, ranging from 10 to 12 months [3] and no significance was noted between two groups (P=0.999), the result may be different if longer follow-up duration for the same population in the 15-week group was allowed in the future.

Particularly in the 25-week group, the administration of CHOP or CMOP didn’t result in significant differences in the response rate, median TTP (242 days versus 273 days) and OST (313 days versus 379 days). Similar findings were noted in the investigation by Wang et al. [49] as well, where CHOP or CMOP were given in each 22 dogs, resulting in median TTP of 222 days and 162 days (P=0.75) and median OST of 318 days and 242 days (P=0.63) , respectively.

5.2 Adverse events

In the present study, most of the adverse events were low-grade toxicities and generally well-tolerated, similar to previous findings. Although the incidence of adverse events among different studies were variable and couldn’t be actually compared due to

common. In terms of grading of toxicities, generally more than half of episodes fell between grade 1 to 2 in most studies [1, 24-26, 49, 52, 53], regardless of different multiagent chemotherapy protocols administered. Besides, only in protocols involving co-administration or escalated dose of chemotherapeutic agents were significantly greater treatment-related toxicity found. Higher frequencies of treatment delay and dose adjustment were accompanied as well [24, 28, 30]. In the study by Chun et al., 35% of population in high-dosed group experienced treatment delays, where dosage of cyclophosphamide and doxorubicin was increased to 250 mg/m² and 37.5 mg/m² respectively, while 43% required dose adjustments. Also, higher percentage of severe hematologic and gastrointestinal toxicity was noted, with incidence of 24% and 16%

respectively [28]. In the study by Burton et al., 62.7% of adverse events occurred after co-administration of vincristine and cyclophosphamide, compared to 1.3 to 18.7% after other single agent only from the same protocol [24]. These findings demonstrated that both designs of protocol from the present study were not expected to result in intolerable toxicities during treatment due to no adoption of aggressive regimen strategies such as dose escalation and co-administration of chemotherapeutic agents.

Sterile hemorrhagic cystitis (SHC) was noted in 2 (4.8%) dogs from the 25-week group and 1 (4.5%) dog from the 15-week group after first, second and third administration of cyclophosphamide, respectively. Currently identified risk factors for developing sterile hemorrhagic cystitis include younger age at diagnosis, longer length of induction protocol and higher cumulative cyclophosphamide dose [54]. The prevalence of SHC was estimated to be 3.8% according to a large pharmacy database [54], where a total of 583 dogs with lymphoma prescribed with oral cyclophosphamide were identified. Furthermore, the prevalence was also found to be associated with the number of cyclophosphamide cycles according to the same database, with 2.8% for

dogs receiving fewer than 10 cycles of cyclophosphamide treatments and 9.1% for dogs administered with 10 or more cycles. The incidence of SHC in the present study seemed to be higher based on the number of administration suggested above. Possible explanation included that the dosage of cyclophosphamide given in NTUVH generally started from 250 mg/m² which was mildly higher than the dosage (200 mg/m²) from the protocol used in part of the dogs from that database, probably resulting a higher cumulative dose here even under the same total cycle of drug administration.

5.3 Prognostic factors

There have been several well-known prognostic factors in canine multicentric lymphoma. Stage V, substage b, T-cell immunophenotype, and presence of thrombocytopenia at diagnosis are factors commonly identified to be associated with both shorter TTP and OST [33-36, 38, 40, 55-57]. Presence of anemia at diagnosis and pretreatment with steroid are more related to shorter survival time [35, 56, 58].

Factors associated with achieving CR or not are less commonly investigated, probably due to the fact of generally high response rate to treatment as a nature of lymphoma. Only in a recent and large-scaled study by Childress et al. in 2018 were factors associated with response mainly analyzed. Presence of thrombocytopenia at diagnosis, higher baseline serum globulin concentration, and greater age at diagnosis were more likely to have partial remission in 98 dogs investigated [36]. In the present study, however, these factors were not identified. Instead, other than body weight and breed which would be discussed later, thoracic involvement in imaging at diagnosis was showed to related to a lower rate of CR (100% versus 76.9%, P=0.018), with dogs

nodes) in thoracic less likely to achieve CR. Abnormalities in thoracic cavity may indicate a more extensive disease involvement or result in a poorer clinical performance at diagnosis. In some patient, respiratory clinical signs were also accompanied and led to the designation of substage b [59], a frequently reported factor associated with poorer prognosis. Due to the fact that the exact causes of the abnormal imaging findings was not routinely further confirmed in these dogs, there was possibility of causes other than lymphoma infiltration accounting for the same imaging finding. Also, the imaging information was only available in 47 dogs (21 dogs in the 25-week group and 26 dogs in the 15-week group), making the sample size smaller and the statistical power less when analyzing this subgroup. Therefore, interpretation of the result here should be taken cautiously.

In univariate analysis of prognostic factors associated with treatment response (CR or not), median TTP and median OST, dogs with body weight higher than median value and Golden retriever were both found to have significantly lower rate of CR and shorter TTP and OST. However, to the author’s knowledge, both factors haven’t been recognized as negative factors for canine multicentric lymphoma in any previous studies.

On the other hand, although Golden retriever has been recognized as a breed well known for lymphoma development and a lifetime risk of nearly 1 in 8 was reported [7, 60], it hasn’t been reported as a poor prognostic factor either. Besides, for canine multicentric lymphoma, no breed has been shown as a poorer responder yet.

Interestingly, lack of significance for these two factors was showed by multivariate analysis for TTP and OST, which possibly meant that body weight and breed in the present study were two confounding factors with interaction between each other existed.

It wasn’t difficult to see the correlation that the population with higher body weight would be majorly comprised by large breed dogs such as Golden retrievers. So basically,

there was probably a single population, mainly Golden retrievers, from the present study accounting for the presence of these two factors at the same time. Why this phenomenon was able to result in a statistically poorer outcome was speculated to be associated with two reasons. First, inaccuracy for body surface area (BSA) tended to be noted in individuals at extremes of weight. At this point, not only Golden retrievers and other large breed dogs were involved, but also obese patients. Although scarce information in veterinary medicine, the impact of extreme height and weight on BSA has been investigated in human medicine. It was found that dosing differences based on BSAs calculated by different formulas were most apparent when patients were at both higher height and weight [61, 62]. Moreover, for obese patients, it has been recommended to dose based on actual body weight instead of making ideal body weight-based empirical dose reduction, according to the finding that concerns about toxicity or overdosing based on the use of actual body weight was not found [63]. In the present study, some of the obese patients or large breed dogs were dosed by the BSAs with empirical reduction at initiation of treatment or during treatment when patients gaining weight (data not showed). Therefore, the efficacy of maximum-tolerated dose (MTD) chemotherapy may be interfered due to underestimation of BSA and subsequently under-dose in these patients. For the second reason, overrepresentation of Golden retriever in the present study was noted meanwhile. The percentage of Golden retriever was 23.8% in the 25-week group and 27.3% in the 15-week group, comprising 25.6% population in a whole. This percentage seemed to be mildly higher compared to the those of Golden retriever in other studies ranging from 7.3% to 22.7% [1, 24, 26, 28, 30, 49, 52, 53, 64-66], with only 3 studies over 20% [49, 64, 65]. Therefore, the less

statistically significance which hadn’t been seen previously. When these confounding factors were controlled in multivariate analysis, only response to treatment remained significant in median TTP. On the other hand, we recognized the population with larger size (i.e. over 20 kg) from others, and further compared Golden retrievers and

Response to treatment (CR versus non-CR), as mentioned above as the only factor remaining significant in multivariate analysis for TTP, has been identified as a prognostic factor in other researches as well [1, 28, 30, 40]. In the study by Jagielski et al. in 2002, where 63 dogs were included, both significantly longer first remission duration and survival time (215 days versus 75 days and 266 days versus 129 days, respectively) were noted for dogs achieved CR [40]. In the initial research studying the 15-week protocol by Curran et al. [1], experiencing a CR as best response was also found to have a positive prognostic influence on both TTP (202 versus 41 days, P<0.0001) and OST (331 days versus 110 days, P=0.0003) in 134 dogs. In their study, this factor remained significant only for TTP rather than OST in multivariate analysis, a similar finding as our study. Based on this point, it was crucial to achieve CR as much as possible during the treatment. Even though multicentric lymphoma was a systemic disease, control of grossly measurable disease to the most extent still seemed to be beneficial for prolonging disease-free status and improving quality of life. As for OST, there were variable factors possibly influencing the outcome as well, such as the use of rescue protocol, the impact of delayed treatment-related toxicity from initial treatment

(e.g. cumulative cardiotoxicity induced by doxorubicin) on clinical performance and owner’s decisions, all making OST more easily biased than TTP in terms of efficacy evaluation of treatment. Accordingly, the effect of complete responder may not be able to show in OST.

The prognostic value of presence of treatment-associated adverse events had been identified in several studies, regardless of different chemotherapy protocols administered [1, 24, 30, 67]. Prolonged first remission duration was found in dogs either developing at least one grade III or IV neutropenia [67], or requiring hospitalization for adverse events [1]. Longer TTP and OST were noted in dogs requiring treatment delays or dose reductions during treatment than those who didn’t [24, 30]. In the present study, the presence of neutropenia during treatment was associated with significantly prolonged OST (514 days versus 309 days, P=0.029) in univariate analysis. However, further analysis of the presence of either at least one grade III or IV neutropenia showed no statistical significance (P=0.069 and 0.253, respectively). Lack of significance in subgroups of more severe grading of toxicity in the present study may be related to the low number of dogs experiencing and episodes happened (17 dogs with 16 episodes for grade III neutropenia and 8 dogs with 9 episodes for grade IV neutropenia), contrary to the study by Vaughan [67] where 40 instances and 35 instances for grade III and IV neutropenia respectively were included in the analysis. The statistical power may be low here. For dogs experiencing adverse events, higher drug exposure was also expected, based on the assumption that there should be variation of drug metabolism and clearance between individuals [1]. For dogs without toxicity presented, the effective dose for tumor may be not reached and therefore less favorable outcomes in this

able to mirror systemic exposure and serve as an indirect parameter for monitoring the responsiveness of tumor to dose given. Similar findings had been noted in several human studies in adjuvant chemotherapy of breast cancer [68-71]. Tailored dose modification according to toxicity may enable the achievement of maximum tolerated dose in individual. However, for dose escalation, standard of care had been inconclusive and not extensively explored in small animal. In human, there was a few studies focusing on the feasibility of neutrophil-guided dose escalation [72-75], with 10-25%

escalation for patients without grade 3 and 4 neutropenia after standard-dosed treatment.

The use of primary granulocyte colony-stimulating factor (G-CSF) support was sometimes involved, but increased risk of adverse events and myelodysplastic syndrome was also accompanied [72]. For small animal, currently no species-specific G-CSF product was available either. Therefore, for neutrophil-guided dose escalation without G-CSF support, the study by Drooger et al. and Edlund et al. showed both acceptable toxicity and feasibility. Notably, efficacy following these dose adjustments however was not mentioned. Fortunately, an ongoing trial (CHOP Dose Escalation for Canine Lymphoma) in Flint Animal Cancer Center of Colorado State University is recruiting canine multicentric lymphoma patients. They are going to assess the ability to successfully escalate drug doses in dogs treated with the 15-week CHOP chemotherapy using a standardized dose escalation protocol, and the effect on outcome and toxicity will be evaluated as well. Their results should be able to provide more information regarding the above issue in the future.

Other factors well-known to be prognostic, such as stage V, substage b, T-cell immunophenotype, thrombocytopenia and anemia, were not recognized in this study.

For clinical stage, bone marrow biopsy was not performed in any dogs from this study, making the designation of stage V less confident. As previously reviewed, the status of

peripheral blood may not be able to sensitively and specifically reflect that of bone marrow in the aspect of lymphoma infiltration according to the findings of flow cytometry and PCR [38, 39]. Therefore, dogs actually with bone marrow involvement but not showing abnormal findings in peripheral blood yet may be false-negatively staged as stage IV (or lower), making potentially less favorable outcomes mingled within those from dogs with true stage IV. Ultimately, the difference in outcomes between stage V and other stages turned less evident.

Substage in the present study, although was almost showed significant (P=0.069) in univariate analysis of TTP, was still not prognostic. For substage, there is no quantitative or objective criteria for determination. Inter-individual variation in substage determination due to subjective assessment of severity and different criteria adopted should be taken into consideration. In the study by Barber et al., a questionnaire was devised to ascertain the clinical parameters and attributes veterinary oncologists considered when assigning clinical substage designation to dogs with multicentric lymphoma [59]. Gastrointestinal, constitutional and respiratory signs were the most commonly identified clinical factors, with greater than 90% respondents indicating that inappetence, vomiting, diarrhea, changes in attitude, weakness and dyspnea were integral in assigning clinical substage. Metabolic, neurologic and nutritional parameters, however, were also considered in more than three-quarters of respondents. In terms of severity, mild-to-moderate severity of signs was reported to be sufficient for substage b designation. For studies in the future, by referring the parameters and criteria discussed above, synchronizing personal standard for substage designation can be expected, which should be helpful in minimizing the disagreement in substage from subjective

For immunophenotype, unavailability of information in a considerable number of dogs from both groups limited the statistical power. Also, breeds more predispositioned towards T-cell immunophenotype, such as Northern and Asian breeds (e.g. Shi-tzu, Husky and Welsh corgi) [4, 60] only presented in 11% population of this study, making

For immunophenotype, unavailability of information in a considerable number of dogs from both groups limited the statistical power. Also, breeds more predispositioned towards T-cell immunophenotype, such as Northern and Asian breeds (e.g. Shi-tzu, Husky and Welsh corgi) [4, 60] only presented in 11% population of this study, making