Use of lomustine (CCNU) in a case of cutaneous equine lymphoma

Abstract

A 12-year-old gelding was diagnosed with recurrent lymphoma in multiple cutaneous sites. A highly invasive preputial mass caused urethral obstruction. The horse was treated with surgery and chemotherapy consisting of lomustine (CCNU) and prednisolone. The treatment was well-tolerated and effective. This is the first reported use of lomustine (CCNU) in a horse for the treatment of equine lymphoma.

Cutaneous lymphoma is a rare skin tumor and uncommon form of lymphoma, especially in horses. Several treatment options have been reported in the dog but are scarce in the horse. Reports of chemotherapy administration in the horse for any form of lymphoma are limited. This case report demonstrates the first reported use of lomustine (CCNU) in a horse for the treatment of equine lymphoma. To the authors’ knowledge, the use of this chemotherapy in horses has not been reported in the veterinary literature.

Case description

A 12-year-old Appaloosa gelding was referred to the Atlantic Veterinary College Veterinary Teaching Hospital (AVC-VTH) for removal of a mass from the inner lamina of the prepuce. The horse had been presented to the AVC-VTH 1 year earlier with a small, subcutaneous mass at the same site. At that time, the mass was surgically removed and diagnosed as cutaneous lymphoma with clean surgical margins. Re-growth of the mass had been noted 4 mo prior to the 2nd admission but the mass had grown rapidly over the previous month and the horse could no longer retract the penis. An additional mass had been noted on the right side of the neck by the owner but was attributed to trauma.

On physical examination, a 3 cm × 3 cm subcutaneous mass was noted on the right aspect of the neck. Examination of the previous surgery site revealed phimosis and a 7 cm × 6 cm × 4 cm, ulcerated mass on the dorsal aspect of the penis, as well as 2 smaller masses located distally along the prepuce of the penis (Figure 1). There was evidence of urine scalding on the ventral abdomen and observation of urination revealed dysuria and “spraying” of urine in an abnormal stream. Due to recurrence and local invasiveness of the preputial tumor with potential partial urethral obstruction, phallectomy was recommended.

Figure 1.

Penis and prepuce of 12-year-old gelding prior to surgery. The head is to the left and tail to right. Note the large ulcerated mass proximally within the external fold of the prepuce and 2 smaller masses distally within the internal fold.

Pre-operative complete blood (cell) count (CBC) and serum biochemistry were within normal limits with the exception of a mild normocytic, normochromic anemia [hematocrit 0.291 L/L, reference range (RR): 0.32 to 0.52 L/L, hemoglobin 101 g/L, range: 110 to 190 g/L]. The day following admission, the horse was administered penicillin G sodium (Novopharm; Toronto, Ontario), 22 000 IU/kg body weight (BW), IV, gentamicin (Gentocin; Schering-Plough, Kirkland, Quebec), 6.6 mg/kg BW, IV, and phenylbutazone (Vétoquinol, Lavaltrie, Quebec), 4.4 mg/kg BW, IV and anesthesia was induced with xylazine (Xylamax; Bimeda, Cambridge, Ontario), 0.5 mg/kg BW, IV, butorphanol tartrate (Torbugesic; Wyeth, Guelph, Ontario), 0.02 mg/kg BW, IV, diazepam (Sandoz, Boucherville, Quebec), 0.04 mg/kg BW, IV, and ketamine hydrochloride (Vetalar; Bioniche, Belleville, Ontario), 2.2 mg/kg BW, IV, and maintained with isoflurane (PCC; Richmond Hill, Ontario) and oxygen. William’s technique of penile amputation was performed as previously described (1) and resected tissues were submitted for histopathological examination. Fine-needle aspirate of the subcutaneous neck mass was also obtained at the time of surgery and submitted for cytology. Recovery from surgery was uneventful and the horse was maintained on penicillin G sodium, 22 000 IU/kg BW, IV, q6h, and gentamicin, 6.6 mg/kg BW, IV, q24h, for 72 h after which the antibiotic regimen was changed to sulfamethoxazole and trimethoprim (TMS) (Apo-Sulfatrim-DS; Apotex, Toronto, Ontario), 25 mg/kg BW, PO, q12h for 14 d. Pain management was achieved with butorphanol tartrate, 0.02 mg/kg BW, IM, q4h, and phenylbutazone, 4.4 mg/kg BW, IV, q12h for 48 h, and then phenylbutazone, 2.2 mg/kg BW, PO, q12h, alone for 10 d.

The amputated penis was submitted for histopathology. Several, multi-nodular, solid, firm subcutaneous masses located within the subcutis were examined. There was extensive ulceration of the overlying epidermis. Microscopically, the masses were composed of locally invasive, poorly defined, dense sheets of neoplastic round cells supported by minimal fine fibrovascular stroma. The predominant small dense round cell populations had round to oval nuclei which were generally uniform in size and measured 5 to 7.5 μm in diameter. These tumor cells had coarse chromatin, small to unapparent nucleoli, and sparse cytoplasm. A sparsely scattered population of slightly larger scattered round cells with nuclei ranging from 10 to 12.5 μm in diameter was also present. Occasionally these cells had large, rarely convoluted or lobulated nuclei or binucleated cells were present. Anisokaryosis was generally moderate. Mitotic figures were common and mainly involved the smaller cell population (8 to 10 per 10 high power field). Scattered small foci of lytic necrosis were noted rarely (Figure 2A). Neoplastic infiltrates abutted the overlying epidermis in one area near the location of skin ulceration but otherwise tumor cells were separated from the epidermis by a layer of normal dermis. Minimal infiltration of the surface epithelium was noted. These findings were compatible with a diagnosis of non-epitheliotropic lymphoma. With immunohistochemical staining, most of the tumor cells stained positively with CD3 markers favoring a T-cell origin. The sparse population of larger atypical round cells stained positively for CD20 (Figures 2B, C). These positive-staining cells may have represented reactive B-cells but the possibility of a T-cell rich B-cell lymphoma (TCRBCL) could not be completely ruled out. Tumor cells were noted close to, but not at, the surgical margins of the phallectomy site. Cytology of the subcutaneous neck mass was also consistent with a lymphohistiocytic infiltrate and lymphoma was suspected.

Figure 2.

Histopathology and immunohistochemistry of penile masses. A — Highly cellular infiltrates of mixed lymphoid cells consisting predominantly of small monomorphic lymphocytes with few scattered larger, more pleomorphic round cells (H&E ×20 objective). Few scattered larger lymphoid cells are present that have lymphohistiocytic morphology. B — scattered, moderately dense aggregates of CD20 positive B-cells among the non-staining T-cells (CD20 immunohistochemical stain, ×20 objective); C — the majority of lymphoid cells are T-cell in origin (CD3 immunohistochemical stain, ×20 objective)

Because lymphoma was present at multiple sites, the inability to rule out other internal masses due to diagnostic limitations in horses, and questionable surgical margins, a systemic chemotherapy protocol was investigated. As CCNU has been used in dogs with cutaneous lymphoma, the canine protocol was adopted and a dose was calculated. The horse was discharged from hospital 12 d after admission to allow the surgical sites to heal completely before beginning the chemotherapy protocol.

Four weeks after discharge, however, the horse was re-presented to the AVC-VTH on emergency with signs of straining and abdominal pain that the owner attributed to an inability to urinate. Upon admission the horse was bright, alert, and responsive, and comfortable. Rectal palpation revealed a flaccid and small bladder indicating the horse had recently urinated. Examination of the penile stump revealed re-growth of a mass measuring 5 cm × 3 cm × 3 cm. A urinary catheter could not be passed through the urethra beyond the level of the mass. At this time, options discussed with the owner included euthanasia or permanent urethrostomy (PU) and start of the chemotherapy protocol. The owner opted for PU and the horse was administered dexamethasone (Dexamethasone 5; Vétoquinol), 0.55 mg/kg BW, IM, q24h for 48 h in an attempt to decrease urethral inflammation in an effort to pass a urinary catheter to facilitate surgery.

Forty-eight hours later the horse again showed signs of acute urinary obstruction including colic, straining, distress, and an enlarged, turgid bladder on rectal palpation. A urinary catheter still could not be passed beyond the level of the mass. The horse was sedated with a constant rate infusion of detomidine hydrochloride (Dorsmoseden; Pfizer, Kirkland, Quebec), 0.02 mg/kg BW per hour to effect, and epidural anesthesia was performed with lidocaine (Lurocaine; Vétoquinol), 0.2 mg/kg BW and xylazine, 0.2 mg/kg BW. Emergency PU was performed to relieve the urinary obstruction and the urethral mucosa was sutured to the skin with 2-0 polypropylene (Prolene; Ethicon, Somerville, New Jersey, USA), in a simple interrupted pattern to create a permanent stoma. Postoperatively the horse was administered TMS, 25 mg/kg BW, PO, q12h for 14 d, phenylbutazone, 2.2 mg/kg BW, PO, q12h, for 5 d, and omeprazole (Gastroguard; Ancaster, Ontario), 1 mg/kg BW, PO, q24h, for 14 d. There was mild dehiscence at the surgical site but healing of the permanent PU site was adequate. Normograde urethral endoscopy was performed through the PU site 3 d following surgery and revealed occlusion of the distal urethra with a soft tissue mass, beyond which, the endoscope was unable to be passed.

The chemotherapy protocol was begun 5 d after surgery and consisted of CCNU (CeeNU; Bristol-Meyers-Squibb, Montreal, Quebec), 65 mg/m², via nasogastric tube, q30d, and prednisolone solution (Trutina Pharmacy; Ancaster, Ontario), 1 mg/kg BW, PO, q24h. Prior to administration, CBC and serum biochemistry were performed as a baseline for neutrophil and platelet numbers and liver values. The CBC and serum biochemistry were repeated 7 d post-administration of CCNU. A CBC only was performed prior to administration of each subsequent chemotherapy treatment. Four days after beginning the chemotherapy protocol, urine was noted at both the PU site and the distal penile stump. Repeat urethral endoscopy was performed 8 d after beginning chemotherapy. The soft tissue mass was no longer visible on endoscopy and the scope could be passed in retrograde fashion to the bladder. In addition, the mass previously present on the right side of the neck was no longer palpable.

The horse remained hospitalized for 52 d during which nursing care of the PU site, sequential monitoring of CBC, serum biochemistry and mass recurrence occurred, and the next dose of CCNU was administered. Liver values and WBC count remained within reference ranges throughout hospitalization. The horse was discharged to the owner and referring veterinarian with 2 additional doses of CCNU to be administered q30d following normal CBC and serum biochemistry. The horse remained on daily prednisolone therapy for 2 mo beyond the completion of the CCNU protocol and then was weaned off the medication over a 6-week period. Three months after discontinuation of all medication, there was recurrence of a small mass on the distal penile stump. The horse was placed back on low dose prednisolone, 0.05 mg/kg BW, q24h, and the mass regressed. Sixteen months after discharge, the horse was still on low dose prednisolone, urinated from both the PU site and the distal penile stump and was being used for pleasure riding.

Discussion

Lymphoma is one of the most common malignancies in the horse and can occur at any age with no apparent breed or gender predilection (2). There is significant variation in regard to clinical signs and course of disease, diagnostic data, and pathological findings. Classification of equine lymphomas has been determined based on anatomic location, morphology, and immunophenotype. Cutaneous lymphoma is a rare neoplasm of horses and can be divided into nonepitheliotropic and epitheliotropic forms (3). Epitheliotrophic lymphoma, also known as mycosis fungoides, typically presents as multifocal to generalized exfoliative dermatitis and focal areas of nodules or ulceration with or without pruritis. Nonepitheliotropic cutaneous lymphomas involve the dermis and subcutis. Clinical findings include cutaneous lesions that are typically multiple and widespread while others may be more localized. Most horses have concurrent systemic illness but some horses, especially those with the histiolymphocytic phenotype, can have skin lesions for months to years before systemic involvement occurs (3). The immunophenotype for nonepitheliotropic cutaneous lymphoma is usually heterogenous and has been classified as T-cell, B-cell, and T-cell-rich B-cell lymphoma (TCRBCL). The latter form of the disease appears to have a slow clinical course with reported survival times of 5 mo to 11 y (4).

T-cell-rich B-cell lymphoma was first reported by Kelley and Mahaffey (5). They described 11 of their 31 cases of equine lymphoma as having 2 immunophenotypically different lymphoid cell populations. In a 2007 study of 5 horses with malignant cutaneous lymphoma, the authors found that the cutaneous tumors appeared to be of T-cell origin only, which contrasted with previous reports (4,6,7). Recently, 203 cases of equine lymphoma were reviewed and classified via morphology and immunophenotyping according to the veterinary adaptation of the World Health Organization (WHO) classification system (8). Similar to the immunophenotyping in this case report, Durham et al (8) found that TCRBCL was the most common lymphoma subtype in the horse, occurring in 43% of their cases and the most common clinical presentation was multiple subcutaneous nodules.

Although chemotherapy has been used in the horse, reports in the veterinary literature are limited. In 1 case of mixed cell thoracic lymphoma in a mare, a protocol of cytarabine, cyclophosphamide, and prednisolone was successfully used (9). Another protocol consisted of cytarabine, chlorambucil, or cyclophosphamide and prednisolone in 2 pregnant mares with lymphoproliferative disease (10). A horse with a leiomyosarcoma was treated with doxorubicin but had progressive disease (11). Reports of treatment of cutaneous lymphoma are even more rare. Drugs that have been used include oral megestrol acetate and intralesional betamethasone (12); methotrexate and dexamethasone (3); and low dose cyclophosphamide in combination with an autogenous tumor cell vaccine (13). To the authors’ knowledge, there are no reports of the use of CCNU in horses.

Lomustine (CCNU) is a nitrosourea alkylating agent used in veterinary medicine to treat a variety of canine and feline cancers including lymphoma, mast cell tumors, and histiocytic sarcoma. In humans, CCNU has had the most antitumor activity for the treatment of brain tumors and lymphoma. Alkylating agents are antitumor drugs that act through the covalent binding of alkyl groups to cellular molecules. As a class, the alkylating agents share a common target (DNA) and are cytotoxic, mutagenic, and carcinogenic. They differ greatly in their toxicity profiles and antitumor activity (14). The most common toxicities associated with CCNU include severe myelosuppression (specifically, neutropenia), thrombocytopenia, and hepatotoxicity. Prolonged or permanent hypoplasia of the bone marrow may be seen after CCNU administration. The onset of neutropenia and platelet depression sometimes can be delayed and occurs 3 to 4 wk after administration; cumulative bone marrow depression may occur with repeated doses.

The choice to use CCNU as a chemotherapeutic agent in this horse was based on the effectiveness of CCNU in the treatment of canine cutaneous lymphoma. Canine cutaneous lymphoma is an uncommon skin tumor of T-lymphocytes that may be epitheliotrophic or nonepitheliotrophic. Epitheliotrophic T-cell lymphoma is the most common variant of these cutaneous lymphomas and is most often seen in older dogs. The clinical presentation of this tumor is variable and ranges from solitary plaques or nodules to a generalized scaling and erythema (15). The treatment for cutaneous lymphoma varies depending upon the extent of disease and has included surgery, external beam radiation therapy, retinoids (isotretinoin), topical chemotherapy with mechlorethamine, and combination chemotherapy protocols with cyclophosphamide, vincristine, cytosine arabinoside, and prednisone. The most common chemotherapy used to treat canine epitheliotropic lymphoma is CCNU (16).

Risbon et al (17) retrospectively evaluated the efficacy of CCNU in the treatment of canine cutaneous epitheliotrophic lymphoma. Treatment with CCNU was effective in > 80% of the study population that resulted in a response rate and duration that was superior to previous studies on CCNU for treating relapsed lymphoma and similar to another study of CCNU for the treatment of canine epitheliotrophic lymphoma (17,18). The overall median duration of response was 94 days. The dose of 60 mg/m² used in this study was well-tolerated. Increased liver enzyme activity, cumulative thrombocytopenia, and neutropenia were the most common toxicities seen. Median survival times were not reported in this retrospective study. However, in another study reviewing 30 cases of dogs with cutaneous epitheliotropic T-cell lymphoma, median survival times after diagnosis for dogs treated with CCNU, prednisolone, or no treatment were 6, 4.25, and 3 mo, respectively (19).

Reports of treatment for nonepitheliotropic cutaneous lymphoma in dogs and cats are scarce. In a single case report of cutaneous nonepitheliotropic B-cell lymphoma in a golden retriever dog, the dog was treated with prednisolone alone (20). The dog was euthanized 6 mo later with marked enlargement of the skin nodules, severe anemia, mild lymphopenia, lameness, and enlarged peripheral lymph nodes. The use of CCNU has been reported in the treatment of nonepitheliotropic T-cell lymphoma in a cat (21). The cat had severe skin lesions that initially involved only the skin of the nose but later progressed to lesions on the ears, chin, digital pads, digits, and corners of the mouth. Although clinically the skin lesions in this cat resembled those associated with epitheliotropic cutaneous lymphoma, the progression of this tumor was very slow as the lesions had been present for over 2 y. Over that period of time, the cat had been treated with prednisolone and antimicrobials but no improvement was seen. After 3 treatments with lomustine (q21d), all of the skin lesions resolved. Two months after lomustine was discontinued, the cat’s azotemia became severe and the cat died. At the time of death, the cat remained free of pruritic skin lesions.

Chemotherapy is commonly used in dogs and cats, especially for lymphoma, as the response rate is high. However, its use in horses is limited most likely due to cost and potential side effects. For the most part, palliative treatment with corticosteroids is the chosen protocol. Depending on location, horses with cutaneous lymphoma could still have a favorable prognosis as this disease appears to follow an indolent course with waxing and waning of lesions. In this case, initial treatment with surgery alone was successful for 1 y. However, due to location of the tumor which led to urethral obstruction, surgery alone was not an effective treatment for this patient. The combination of CCNU with prednisolone decreased the size of the tumor and the horse was able to urinate through the distal penis 4 d after beginning treatment. It is possible that prednisolone alone may have led to the same result, but single agent prednisolone does not appear to be an effective treatment for nonepitheliotropic lymphoma (20,21). In summary, findings in this horse suggest that CCNU in the adjuvant setting may be useful for the treatment of cutaneous nonepitheliotropic lymphoma in horses and further investigation is warranted. Lomustine appears to be a safe, well-tolerated and cost-effective chemotherapeutic agent for treatment of equine lymphoma.