Introduction

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Lomustine (CCNU), which is an alkylating drug belonging to the family of nitrosoureas, is often used as a first-line medical therapy to treat canine HS. Although clinical benefits have been demonstrated for CCNU treatments in HS dogs, with response rates of 29–46%,^1,2 this drug can cause severe myelosuppression and cumulative dose-related irreversible hepatotoxicity.^3–5 Therefore, accurate dosing is important in dogs. However, because CCNU is administered as an oral capsule (available in 10-mg and 40-mg strengths), it can be difficult to achieve accurate dosing. For this reason, veterinary clinicians often have CCNU compounded by veterinary compounding pharmacies. However, it has been suggested that these compounded CCNU capsules have less CCNU content and thus have a weaker potency with wider range of variability as compared with the FDA-approved CCNU capsules.^6,7 Moreover, CeeNU, which was the FDA-approved form of CCNU, was acquired by NextSource Biotechnology from Bristol-Myers Squibb in 2013 and relaunched under the name of Gleostine. This change in product ownership resulted in a greatly increased drug price, which subsequently impeded the use of CCNU. Thus, investigations into alternatives to CCNU for the treatment of HS would be beneficial.

Nimustine, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), is an alkylating drug that belongs to the same family of nitrosourea compounds as CCNU. In contrast to CCNU, ACNU is water soluble and thus can be used as an IV/arterial injectable drug. As a result, this makes it possible to achieve accurate dosing in dogs compared to the preformed capsule sizes and probably the compounded formulations. ACNU has been demonstrated to exhibit therapeutic activity in various human tumors, such as gliomas, lung cancers, hematopoietic tumors, and gastrointestinal tumors.^8,9 ACNU has also been shown to have the highest potency for inducing deoxyribonucleic acid interstrand crosslinks among the nitrosoureas, including CCNU, carmustine, and fotemustine.¹⁰ Consistent with this mechanism, ACNU has also been shown to have a better survival gain than carmustine, CCNU, and fotemustine in human patients with high-grade glioma.⁹ Moreover, a phase I study of ACNU in tumor-bearing dogs demonstrated acceptable safety and good tolerability of this drug in dogs at a dose of 25 mg/m² with an interval of 3 wk.¹¹

Based on the results of the phase I study and the advantages of ACNU, including the potent deoxyribonucleic acid interstrand crosslinking activity and the IV injectable form, ACNU may be an alternative to CCNU for the treatment of HS. Thus, our current study examined the therapeutic potential of ACNU in dogs with HS.

Materials and Methods

Data were retrospectively reviewed from the medical records of dogs diagnosed with HS that were treated with ACNU (Nidran^a) in the primary as well as adjuvant settings at the Veterinary Medical Teaching Hospital of Nippon Veterinary and Life Science University from 2013 to 2018. Dogs were eligible for inclusion if they had a cytologic and/or histologic diagnosis of HS, had received at least one dose of ACNU, and had sufficient follow-up information.

HS was diagnosed by histopathology or fine-needle aspiration cytology combined with cytochemical staining (positive for α-naphthyl butyrate esterase, which was inhibited by sodium fluoride).¹² In cases with available tissue samples, immunohistochemical staining was performed using various combinations of markers for monocytes or macrophages, including a combination of major histocompatibility complex (MHC) class II, Iba-1, CD163, and CD204 or MHC class II alone.^13,14

Before ACNU treatment, every dog in this series underwent a complete physical examination, blood tests including complete blood count (CBC) and serum chemistry, and imaging tests including abdominal ultrasound and thoracic radiography. No dog had a urinalysis. Advanced imaging tests (computed tomography and/or MRI) were performed as needed. If regional lymph nodes were palpable, metastatic lesions were suspected, or pleural effusion was seen, the presence of HS cells was cytologically or histopathologically examined.

Both physical and CBC examinations were performed weekly, and physical and CBC examinations with serum chemistry and imaging tests (abdominal ultrasound and thoracic radiography) were conducted on the day of each treatment in all of the dogs. These examinations were then performed every 1–4 mo thereafter until the dogs died or were lost to follow-up.

ACNU (25 mg per vial) was dissolved in 5 mL water for injection and intravenously administered (bolus). The intended starting dose and dosing interval were 25 mg/m² and 3 wk, respectively, according to the recommendations of a published phase I study of ACNU in dogs¹¹ except for cases that were treated before publication of the phase I study. In these cases, ACNU treatment was started at a dose of 20 or 30 mg/m² with a 3-wk interval. In the adjuvant setting, treatment was scheduled as five or more doses of ACNU. The treatment baseline requirements were set as neutrophil counts of ≥2500/µL and platelet counts of ≥100 × 10³/µL.

Overall survival (OS) was calculated from the date of the initial ACNU treatment until death. Progression-free survival (PFS) was calculated from the date of the initial ACNU treatment until tumor progression or death from any cause. Tumor progression was defined as worsening or appearance of tumor-associated clinical signs (e.g., pain, neurological signs, and pleural effusion) or a relapse of the tumor. The Kaplan-Meier product limit method was used to calculate median OS and median PFS. For this analysis, the GraphPad Prism software program^b was used.

Toxicities were graded according to the Veterinary Co-operative Oncology Group-Common Terminology Criteria for Adverse Events version 1.1.¹⁵

Results

Table 1 shows the patient characteristics and disease status for the 11 cases that were included in this study. The dog breeds examined included French bulldog (n = 2), Labrador retriever (n = 2), miniature schnauzer (n = 2), flat-coated retriever (n = 1), standard poodle (n = 1), Maltese (n = 1), Shetland sheepdog (n = 1), and mixed-breed dog (n = 1). The median age of the dogs was 9 yr (range, 6–14 yr), with a median bodyweight of 12 kg (range, 4.7–43.1 kg). In 5 out of 11 cases (Cases 1–5), there was no prior antitumor treatment and the animals exhibited gross disease with tumor-associated clinical signs. The remaining 6 cases (Cases 6–11) had been previously treated by surgery or surgery with radiotherapy and exhibited neither gross disease nor tumor-associated clinical signs. Except for cerebral HS cases (Cases 8 and 9), all of these cases had tumor cell–free surgical margins. Tumors of the cerebral HS cases were incompletely excised and Case 8 underwent radiotherapy (48 Gy in 12 fractions, 3 fractions per wk). None of the dogs received chemotherapy prior to ACNU.

TABLE 1 Patient Characteristics and Disease Status

View Fullscreen

Diagnosis was determined based on the histopathology evaluations in eight cases (six cases had immunohistochemistry results) or on the cytology results combined with cytochemical staining using α-naphthyl butyrate esterase in three cases. The diagnostic results are summarized in Table 2.

TABLE 2 Diagnosis of Histiocytic Sarcoma

View Fullscreen

Table 3 summarizes the ACNU treatment conditions and outcomes. ACNU treatment was conducted in a primary setting for five dogs with macroscopic disease (Cases 1–5) and in an adjuvant setting for six dogs (Cases 6–11). Treatments in all dogs started with a median ACNU dose of 25 mg/m² (range, 20–30 mg/m²). The ACNU dose was reduced by 25% in four cases after the second cycle administration because of neutropenia (Cases 4, 6, 7, and 8). Among these cases, in one case (Case 6), the dose of ACNU was re-escalated up to 22.5 mg/m²; no neutropenia occurred at this dose. No other dose escalation was performed in the remaining cases. Dosing intervals ranged from 3 to 5 wk with the number of administrations ranging from one to eight. The median number of administrations was two times in the primary setting and 6.5 times in the adjuvant setting. No case had delayed administration because of side effects. The treatment period ranged from 1 to 218 days. The median period of treatment was 29 days in the primary setting and 112 days in the adjuvant setting. ACNU treatment was terminated because of death (Cases 1, 2, and 5), relapse/tumor progression (Cases 3, 7, and 8), no improvement in pleural effusion (Case 4), surgical wound suppuration (Case 9), or completion of scheduled administration (Cases 6 and 10). Case 11 was still undergoing ACNU treatment at the time of data analysis. During the ACNU treatment period, prednisolone was also administered to Cases 1, 2, and 5, at a dose of 2 mg/kg per os (PO) once daily, 2 mg/kg PO once daily (with taper), and 1 mg/kg PO once daily, respectively.

TABLE 3 Nimustine Treatment Condition and Outcome

View Fullscreen

Case 4 was additionally treated with vincristine, L-asparaginase, mitoxantrone, and toceranib after two administrations of ACNU owing to the lack of improvement in pleural effusion; however, no improvement in pleural effusion as well as no other favorable clinical response was obtained with any of these agents. In Case 6, the tumor relapsed in the skin (multiple and different sites than the primary cutaneous tumor; HS was confirmed histologically in one tumor and cytologically in other tumors) 277 days after completion of the five scheduled adjuvant ACNU administrations (382 days from the initial treatment with ACNU). After tumor relapse, this case was re-treated with ACNU (five additional administrations).

The median OS and PFS for the five dogs treated with ACNU in the primary setting were 120 days (6–173 days) and 63 days (range, 6–131 days), respectively, whereas the values for the six dogs treated in the adjuvant setting were 400 days (>92 to >1286 days) and 212 days (range, >92 to 1286 days), respectively. A total of eight dogs (Cases 1–8) were suspected to have died as a result of tumor progression, and one dog (Case 10) died of unrelated disease (biliary obstruction without evidence of tumor relapse: occurred 105 days after final administration of ACNU). Two dogs (Cases 9 and 11) were still alive at the time of data analysis.

Among the five cases treated with ACNU in the primary setting (Cases 1–5), two cases showed improvement in tumor-associated clinical signs (Cases 2 and 3). These two cases had neurological signs (Table 1) that were caused by spinal cord compression by the tumor (confirmed by MRI in Case 2 and MRI/computed tomography in Case 3). In Case 2, disappearance of dysuria and major improvement in dysstasia were noted 3 wk after the initial ACNU treatment. This physical state was maintained for 99 days. In Case 3, disappearance of dysuria and dysbasia along with improvement in osteolytic pain were noted 3 wk after the initial ACNU treatment. This physical state maintained for 110 days. There was no evidence of improvement in the tumor-associated clinical signs noted in the three other cases (Cases 1, 4, and 5). In Case 6, all of the HS tumors that relapsed after completion of the adjuvant ACNU treatment had disappeared on day 25 and relapsed on day 64 of the second round of ACNU treatment.

The major toxic effect associated with ACNU treatment was neutropenia. In 8 out of 11 cases, although mild to severe neutropenia (grade 1, Case 3; grade 2, Cases 2 and 10; grade 3, Cases 7 and 8; grade 4, Cases 4, 6, and 11) was observed, the dogs did not exhibit any febrile neutropenia. No thrombocytopenia was noted. There were three cases with mild and self-limiting gastrointestinal symptoms, including vomiting (grade 2, Case 10) and anorexia (grade 1, Cases 7 and 10). Fatigue was observed in two dogs (grade 1, Cases 7 and 9). There was no evidence of hepatic toxicity in any of the cases.

Discussion

Our current study demonstrated that the median OS was 120 days (median PFS, 63 days) in the primary setting and 400 days (median PFS, 212 days) in the adjuvant settings. Previous studies investigating the treatment of HS with single-agent CCNU (or with combination chemotherapy protocols containing CCNU) reported median survival times ranging from 106 to 185 days; most of the patients in these studies had gross disease when treated with CCNU.^1,2,16,17 In the adjuvant setting, the reported median survival time for dogs with HS that received CCNU was 568 days,¹⁸ whereas that for dogs receiving CCNU-containing chemotherapy protocols was 219 days.¹⁹ In our case series, four dogs had primary pulmonary HS. In primary pulmonary HS, the median survival times for CCNU-based chemotherapy only and surgery with adjuvant CCNU-based chemotherapy were 131 days (median PFS 91 days) and 374 days (median PFS 276 days), respectively.²⁰ Another study has reported that the survival data for periarticular HS might exhibit a better outcome compared with other types of HS.²¹ However, this type of HS was not included in our current study. Although these studies cannot be directly compared, the outcome of patients treated with ACNU does not appear to be different from those treated with CCNU or CCNU-containing protocols.

In our current study, two dogs (Cases 2 and 3) showed improvement in tumor-associated clinical signs after ACNU treatment. Case 2 was treated with concurrent prednisolone and ACNU owing to neurological signs associated with a spinal tumor. However, it has been reported that corticosteroids may not necessarily have specific antineoplastic activity against HS.^1,22 Moreover, HS that involves the central nervous system may be more aggressive in nature,²³ and this case showed a long cessation period (120 days) of the clinical signs in this case. Case 3 was treated with a nonsteroidal anti-inflammatory drug (piroxicam) because of osteolytic pain and neurological signs that were likely associated with spinal metastasis. However, this treatment was terminated 2 wk prior to the start of the ACNU as a result of a lack of improvement in these signs. Additionally, no concurrent medication was administered in this case. The improvement of the clinical signs may be primarily associated with the ACNU.

In cases treated with ACNU in the primary setting (Cases 1–5), objective tumor responses were not assessed because precise size monitoring was difficult to perform owing to the insufficient size and/or situations of the tumors. However, after subcutaneous tumor relapse following the completion of adjuvant ACNU treatment in Case 6, the HS masses (the largest mass was 17 mm in diameter) subsequently disappeared after retreatment with ACNU. Even though this was just a single case, these findings directly suggest that ACNU may have an antitumor potential in HS.

The major toxic effect of ACNU was neutropenia. In addition, mild gastrointestinal toxicity was observed. No hepatic toxicity was experienced in our current cases. These findings are consistent with the results of a previous phase I study of ACNU in tumor-bearing dogs.¹¹ In terms of hepatic toxicity, ACNU might exhibit different properties as compared with CCNU. ACNU appears to be both safe and well tolerated in dogs with HS.

Because of the retrospective nature, there were many limitations for our current study. These included the small number of cases with heterogeneous disease type/status and the nonstandardized treatment regimen. One of the dogs in our current study had splenic involvement. A previous study has suggested that this may be a negative prognostic factor.¹ The central nervous system was involved in four cases, with the prognosis for this type of HS previously reported to be poor.²³ In the current study, six cases were started on ACNU at a dose of 25 mg/m², which is the dose that was recommended in the phase I study of ACNU in dogs.¹¹ However, five other dogs were started at different dose levels. Moreover, in one case the dog was treated with other chemotherapeutic drugs after the initial treatment with ACNU. Furthermore, the lack of an objective tumor response assessment and inconsistency of the diagnostic approach were also limitations of our current study. Thus, it should be noted that these conditions could have potentially overemphasized or underestimated the current results of our study.

Conclusion

ACNU was well tolerated and showed a similar outcome to that seen for CCNU in terms of OS and PFS in the current study population. A larger prospective and randomized evaluation will need to be undertaken in order to determine if ACNU is an appropriate alternative to CCNU in the treatment of HS.