Rabacfosadine for naïve canine intermediate to large cell lymphoma: Efficacy and adverse event profile across three prospective clinical trials

Abstract While current lymphoma therapies induce remission in most dogs, drug‐resistant relapse is common, creating a need for novel agents. Rabacfosadine (RAB), a double prodrug of the acyclic nucleotide phosphonate 9‐(2‐phosphonylmethoxyethel) guanine (PMEG), preferentially targets lymphoma cells with reduced systemic toxicity compared with PMEG. Previous studies evaluating RAB administered every 21 days have suggested efficacy in both naïve and relapsed subjects; however, no large studies of RAB as a single agent have been reported in previously untreated dogs with intermediate to large cell lymphoma. The purpose of this study was to evaluate the safety and efficacy of RAB in dogs with previously untreated (excluding corticosteroids) lymphoma. Sixty‐three dogs received up to five RAB treatments every 21 days (16 at 0.82 mg/kg and 47 at 1.0 mg/kg) as a 30 minutes intravenous infusion, with (n = 23) or without (n = 40) concurrent corticosteroids. Response assessment and adverse event (Ae) evaluation were performed every 21 days via Veterinary Cooperative Oncology Group (VCOG) criteria. The overall response rate was 87% (52% CR, 35% PR). The overall median progression free interval was 122 days (199 for CR, 89 for PR and 153 days for all responders). T‐cell immunophenotype and corticosteroid pre‐treatment were predictive of inferior outcomes on multivariate analysis. AEs were most commonly of gastrointestinal origin (hyporexia/diarrhoea) and generally resolved with supportive treatment and/or dosage adjustment. Three dogs experienced VCOG‐CTCAE grade 5 delayed pulmonary fibrosis. In conclusion, RAB administered every 3 weeks is generally well tolerated and demonstrates substantial antitumour activity in dogs with previously untreated intermediate to large cell lymphoma.


| INTRODUCTION
Lymphoma, specifically diffuse large cell B-cell lymphoma and peripheral T-cell lymphoma (PTL), is one of the most common cancers in dogs. 1,2 The current standard of care treatments consist of multi-agent chemotherapy regimens. Most commonly, oncologists rely on doxorubicin (DOX)-based protocols [eg, a cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), prednisone (CHOP)-based protocol]. With this approach, multiple drugs are given weekly to biweekly, generally over the course of 19 to 26 weeks. 1,[3][4][5] The frequency of veterinary visits associated with this intensive treatment protocol can become burdensome for some owners and is impossible for others. Less intensive protocols, where cytotoxic drugs are given every 3 weeks, offer a less stringent treatment option, but with the potential risk of shorter remission durations and survival times [6][7][8] ; however, for many owners, this is a reasonable compromise.
Rabacfosadine (RAB; Tanovea-CA1; also referred to as VDC-1101 or GS-9219) is a double prodrug of the acyclic nucleotide phosphonate 9-(2-phosphonylmethoxyethel) guanine (PMEG), that preferentially targets lymphoma cells with reduced systemic toxicity compared with PMEG. 9 It was conditionally approved by the U.S. Food and Drug Administration for the treatment of canine lymphoma in 2016. According to its label, it may be given every 21 days for up to five treatments to dogs with lymphoma of any type. Studies evaluating RAB in both treatment-naive and relapsed or treatment-refractory dogs with lymphoma have reported overall response rates of approximately 50% to 100%, with higher response rates and longer response durations observed in dogs with Bcell lymphoma and those that are less heavily pre-treated. [9][10][11][12] For the most part, RAB has been well tolerated, typically associated with low grade or mild adverse events (AEs) including neutropenia and gastrointestinal (GI) signs. The dermatologic AE associated with RAB often manifests as pruritic otitis externa and/or erythemic skin lesions on the dorsum and in the inguinal areas. These dermatological AEs typically resolve with supportive therapy and/or dose reductions and/or delays. [9][10][11]13,14 Additionally, pulmonary fibrosis is a reported toxicity associated with RAB administration. Although reported in approximately 4% of RAB-treated dogs, it is potentially life-threatening. The mechanism of this AE is not understood, but it appears to be idiosyncratic. Careful monitoring with thoracic radiographs for evidence of pulmonary pathology is encouraged. [9][10][11]13,14 The purpose of this report was to describe the safety and efficacy of was strongly recommended but not required. Similarly, post-mortem examination of dogs that died while on study was encouraged but not required. Only data from dogs with treatment-naïve lymphoma were extracted, with the caveat that dogs receiving prior corticosteroids were considered treatment naïve. Information regarding the type, duration of treatment, and dose of corticosteroids was not readily available, rather this variable was recorded as "yes" prior treatment with corticosteroids or "no" prior treatment with corticosteroids.
Screening tests included physical examination, complete blood count (CBC), serum biochemical profile and urinalysis to ensure dogs met inclusion criteria. Thoracic radiographs were strongly recommended.
Adequate bone marrow and organ function, defined as absolute neutrophil count ≥2000 cells/uL, haematocrit ≥25%, platelet count ≥75 000 cells/uL, creatinine ≤2.5 mg/dL, total bilirubin ≤ the upper limit of normal (ULN), ALT ≤3 times ULN or if >3 times ULN, serum bile acids ≤ULN and a modified ECOG performance score ≤1 were required for inclusion. 8 West Highland white terrier dogs and/or dogs with pulmonary pathology possibly predisposing to fibrosis were excluded. Study protocol approval was obtained from Institutional Animal Care and Use Committees and/or Clinical Review Boards according to individual institutional requirements.
Other treatment options, including no treatment, were discussed with all owners. Owners were allowed to make their own educated decisions about the appropriate choice for their pet, and signed informed consent was obtained from all owners prior to study entry.

| Trial design
RAB was provided by VetDC, Inc. (Fort Collins, Colorado). Depending upon the study (VC-003, VC-007 and VC-010), dogs were treated at 0.82 or 1.0 mg/kg. RAB was reconstituted and diluted with sodium chloride for injection, USP to achieve a total infusion volume of 2 mL/ kg and was administered intravenously (IV) over 30 minutes. Treatments were repeated every 21 days for up to five total treatments. The treatment schedules were uniform across all studies and are outlined in Table 1.
Treatment response was based on measurements (using callipers) of peripheral target lesions using the Veterinary Cooperative Oncology Group (VCOG) Response Evaluation Criteria for Peripheral Nodal Lymphoma. 15 Dogs experiencing complete response (CR) received a total of five RAB treatments; thereafter, monthly rechecks were performed until progressive disease (PD) was noted. Dogs experiencing partial response (PR) or stable disease (SD) after five treatment cycles were considered off-study upon completion of the fifth treatment cycle. Once PD was noted, dogs were removed from the study and were eligible to pursue other treatment(s) as deemed appropriate by the attending oncologist and at the owner's discretion.

| AE assessment
Clinical, haematological and biochemical AEs were assessed based on patient history provided by the owner, physical examination and blood work as outlined in Table TABLE 1 AEs. If a DLT was observed, the dose was reduced by up to 20% for future RAB administrations. Treatment of AEs was undertaken at the discretion of the attending clinician.

| Statistical analysis
Continuous data were expressed as median and range, and categorical data as frequencies and percentages. The objective response rate (ORR) and progression-free interval (PFI) were the primary efficacy endpoints. The ORR was defined as the percentage of evaluable patients experiencing CR or PR as their best response. The PFI was calculated from the date of treatment initiation to the date of PD. Dogs were censored if they were lost to follow-up prior to documentation of PD, if they were withdrawn for a reason other than PD, or if they died of a confirmed cause unrelated to RAB treatment or lymphoma before PD development. Continuous variables were compared between groups of patients using a two-tailed, unpaired T test or Mann-Whitney test depending on data normality, which was assessed using a D'Agostino Pearson omnibus test. Categorical variables were compared between cohorts using a two-tailed Fisher's exact test. The Kaplan-Meier method was used to estimate and display the distribution of PFI. Differences between potential prognostic subsets were compared using logrank analysis. Variables with a univariate P value of <.15 were incorporated into a forward step-

| Cell line validation statement
No cell lines were used in this study.

| RESULTS
Monthly rechecks X X Every other month Abbreviations: CBC, complete blood count; LN, lymph node; PE, physical examination; RAB, rabacfosadine; UA, urinalysis. a Thoracic radiographs were strongly recommended but not required. b If CBC, serum chemistry and urinalysis were performed and evaluated within 7 days of day 0, these were not repeated on day 0. c These were not required for all studies. d This visit was only required in dogs experiencing a dose-limiting toxicity following the first treatment.
(3 dogs), and PD in 8% (5 dogs Reported ORRs, CR rates and median response durations have been 74% to 87%, 52% to 78%, and 80.5 to 169 days, respectively. 6,7,[17][18][19][20] Lomustine and a tapering dose of corticosteroids have also been retrospectively evaluated as first-line treatment in 17 dogs with lymphoma. The reported ORR was 53%, with a median duration of 39.5 days; these results lead the authors to conclude that lomustine should not be used as a first-line therapy for canine lymphoma. 8 Here, we report an ORR of 87% and a median PFI of 122 days for all RAB treated dogs (B-and Tcell). Dogs with B-cell lymphoma had a higher ORR of 97%. It is impossible to compare the results of the current study to those from other independent studies; however, it is noteworthy that our results appear to be within the range of those reported for single agent DOX treatment.
Despite the acceptable results observed with single-agent DOX, all of the aforementioned studies concluded that these single agent treatment protocols are not intended to replace the current standard of care; rather, they simply provide a less time consuming and potentially less costly treatment option. [6][7][8] That being said, drug resistance remains an inevitable consequence of the treatment of canine lymphoma, even when multi-agent protocols are used. 1,[3][4][5]21 The unique mechanism of action of RAB 9 makes it an attractive, novel agent that may be combined with other chemotherapy agents and/or incorporated into CHOP-based protocols in attempt to extend the time to T A B L E 3 Hematologic, gastrointestinal, dermatologic and pulmonary adverse events (AEs) at 0.82 mg/kg (A) and 1.0 mg/kg (B) treatment. If noted, it is best to address these AEs early, as they can be successfully managed with dose reductions and/or delays and supportive medications, rather than discontinuation of RAB. [9][10][11]13,14,29 Pulmonary fibrosis, another AE of RAB, occurred in these studies, at a similar frequency to studies reported previously. 6,9 The mechanism of this remains unknown. The timing of pulmonary fibrosis, including grade 5 pulmonary fibrosis, is also unclear. [9][10][11]13  A potential limitation of the current report is that, although these studies were conducted prospectively, the data were extracted retrospectively. Information regarding the type, duration of treatment and dose of corticosteroids was not readily available, making it difficult to comment further on this significant variable. It is also impossible to say whether or not some of the short-lived responses were simply because of corticosteroid administration; however, concurrent treatment with corticosteroids did not alter the likelihood of response statistically. One could argue that PARR should not be used as a sole test to determine immunophenotype.
Therefore, another limitation could be under-or over-reporting of B vs T cell lymphoma. With that being said, the majority of these cases were immunophenotyped with assays other than PARR. As with previous RAB studies, owners were not asked to keep daily dairies at home to prospectively record any potential AEs, so subtle or mild constitutional and GI AEs (eg, lethargy, hyporexia, vomiting, diarrhoea) may have been underreported. Additionally, target lesion measurement and grading of AEs were performed by a team of clinicians, not necessarily the same clinician at each visit, possibly introducing bias. However, by using the VCOG Response Evaluation Criteria for Peripheral Nodal Lymphoma criteria and the CTCAE, which allow for inter-individual measurement differences and provide specific criteria for AE grading, respectively, we suspect the bias was limited. Finally, necropsies were not required making it difficult to definitively assess causes of death in patients on study.
In conclusion, RAB appears to be an effective treatment for dogs with treatment-naïve, multicentric, intermediate to large cell lymphoma, with an 87% overall response rate and a median PFI of 122 days. The response rate and PFI were longer in dogs with B cell lymphoma; however, half of the dogs with T cell lymphoma also responded to treatment.