Analysis of platinum content in biodegradable carboplatin-impregnated beads and retrospective assessment of tolerability for intralesional use of the beads in dogs following excision of subcutaneous sarcomas: 29 cases (2011–2014)

Theresa A. Hess Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Macy E. Drinkhouse Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Joshua D. Prey Roswell Park Cancer Institute, Elm and Carleton Streets, Buffalo, NY 14263.

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Jonathan M. Miller Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Arthur A. Fettig Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Carol A. Carberry Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Stephen H. Brenn Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Dennis B. Bailey Oradell Animal Hospital, 580 Winters Ave, Paramus, NJ 07652.

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Abstract

OBJECTIVE To evaluate platinum content in biodegradable carboplatin-impregnated beads and retrospectively assess tolerability and outcome data for dogs treated by intralesional placement of such beads following surgical excision of subcutaneous sarcomas.

DESIGN Evaluation study and retrospective case series.

SAMPLE 9 carboplatin-impregnated beads and 29 client-owned dogs.

PROCEDURES Platinum content in 9 carboplatin-impregnated beads from 3 lots was measured by spectrophotometry, and calculated carboplatin content was compared with the labeled content. Medical records were searched to identify dogs with subcutaneous sarcomas for which treatment included placement of carboplatin-impregnated beads between 2011 and 2014. Signalment, tumor characteristics, surgical and histologic data, adverse events, and local recurrences were recorded. Associations between variables of interest and adverse events or local disease-free interval were analyzed.

RESULTS In vitro analysis identified a mean ± SD platinum content of 5.38 ± 0.97 mg/bead. Calculated carboplatin content (10.24 ± 1.84 mg/bead) was significantly greater than the labeled amount (4.6 mg/bead). Bead weight and total platinum content differed significantly among lots, but platinum content per bead weight did not. Mild-to-moderate local adverse events were reported for 11 of 29 tumors; all resolved without additional surgery. No dogs had signs of systemic toxicosis. Overall local disease-free rates 1, 2, and 3 years after surgery were 70%, 70%, and 58%, respectively, as determined by Kaplan-Meier analysis.

CONCLUSIONS AND CLINICAL RELEVANCE Carboplatin-impregnated beads were well tolerated; however, results of in vitro tests indicated that caution is needed because of manufacturing inconsistencies.

Abstract

OBJECTIVE To evaluate platinum content in biodegradable carboplatin-impregnated beads and retrospectively assess tolerability and outcome data for dogs treated by intralesional placement of such beads following surgical excision of subcutaneous sarcomas.

DESIGN Evaluation study and retrospective case series.

SAMPLE 9 carboplatin-impregnated beads and 29 client-owned dogs.

PROCEDURES Platinum content in 9 carboplatin-impregnated beads from 3 lots was measured by spectrophotometry, and calculated carboplatin content was compared with the labeled content. Medical records were searched to identify dogs with subcutaneous sarcomas for which treatment included placement of carboplatin-impregnated beads between 2011 and 2014. Signalment, tumor characteristics, surgical and histologic data, adverse events, and local recurrences were recorded. Associations between variables of interest and adverse events or local disease-free interval were analyzed.

RESULTS In vitro analysis identified a mean ± SD platinum content of 5.38 ± 0.97 mg/bead. Calculated carboplatin content (10.24 ± 1.84 mg/bead) was significantly greater than the labeled amount (4.6 mg/bead). Bead weight and total platinum content differed significantly among lots, but platinum content per bead weight did not. Mild-to-moderate local adverse events were reported for 11 of 29 tumors; all resolved without additional surgery. No dogs had signs of systemic toxicosis. Overall local disease-free rates 1, 2, and 3 years after surgery were 70%, 70%, and 58%, respectively, as determined by Kaplan-Meier analysis.

CONCLUSIONS AND CLINICAL RELEVANCE Carboplatin-impregnated beads were well tolerated; however, results of in vitro tests indicated that caution is needed because of manufacturing inconsistencies.

Soft tissue sarcomas comprise approximately 15% of all cutaneous and subcutaneous tumors in dogs.1 Collectively, these tumors are considered to be very locally invasive, and metastatic rates correspond to tumor grade or, more specifically, mitotic rate.2 When metastasis is not identified, treatment is focused initially on local control of the primary tumor. Surgical resection of the primary tumor with wide margins (≥ 3 cm laterally and ≥ 1 fascial plane deep) is recommended whenever possible.1,3–5 However, depending on tumor size and location, complete surgical excision is not always possible. Reported local recurrence rates for incompletely excised soft tissue sarcomas without any adjuvant therapy range from 4 of 24 (17%) to 55 of 55 (100%).2,5–7 When additional surgery is not possible, adjuvant radiation therapy is usually recommended for incompletely excised soft tissue sarcomas.8,9 Radiation therapy is not always feasible because of variable availability of facilities or financial constraints, and owners can be reluctant to pursue radiation treatment; therefore, other adjuvant treatments have been investigated. Soft tissue sarcomas are considered relatively resistant to chemotherapeutic treatment. Adjuvant doxorubicin treatment did not improve local control, metastatic rate, or survival time in dogs with incompletely excised high-grade soft tissue sarcomas, compared with results for dogs treated by surgery alone.10 In contrast, low-dose oral cyclophosphamide and piroxicam treatment more than doubled the DFI for dogs with incompletely excised low-grade soft tissue sarcomas.7 Metronomic chemotherapy is antiangiogenic, rather than directly cytotoxic to residual tumor cells. Therefore, treatment is continued indefinitely, and patients receiving chronic cyclophosphamide treatment are at risk for cumulative effects such as hemorrhagic cystitis.7

Intralesional chemotherapy takes advantage of the steep dose-response curves for both tumor cell killing and adverse events. An article reviewing the clinical pharmacokinetics of carboplatin indicates that in people who received carboplatin at a dose of 350 mg/m2 of BSA, IV, the mean tissue concentration was 0.22 mmol/L/m2,11 which equates to only 0.02% of the dose reaching each cubic centimeter of tissue. With intralesional therapy, the entire dose reaches the primary tumor site, where it may potentially overwhelm resistance mechanisms and result in a higher rate of tumor cell death. Furthermore, intra-operative placement of a chemotherapy implant allows the dose to be distributed more precisely within the treatment field. A low degree of systemic exposure minimizes adverse systemic effects such as gastrointestinal upset, myelosuppression, and nephrotoxicosis. Intralesional cisplatin and carboplatin have been used in several preparations to treat a variety of tumors in veterinary oncology, including osteosarcoma, soft tissue sarcomas, and oral melanoma in dogs; squamous cell carcinoma of the nasal planum in cats; and soft tissue sarcomas, sarcoids, squamous cell carcinoma, and melanoma in horses.12–20 Previously, 2 cisplatin-containing biodegradable polymer implants were evaluated for the treatment of soft tissue sarcomas in dogs: a liquid polymer solution that solidifies after implantation12 and open-cell polylactic acid sponges.13 However, both of these implants were associated with severe wound-healing complications.12,13 Intralesional chemotherapeutic drugs can also potentially leak from an injection site or through a surgical wound.16,20,21 Postoperative healing complications can potentially increase environmental contamination, placing hospital personnel, pet owners or other persons, and other household pets at risk for unwanted exposure to chemotherapeutic agents.

A new biodegradable implant, a calcium sulfate–dextran sulfate bead containing either cisplatin or carboplatin, is marketed in the United States for use in veterinary patients through a veterinary compounding pharmacy. In an aqueous in vitro model that included complete fluid exchange, the majority of the platinum was eluted over the first 3 days after introduction into PBS solution, but platinum elution was still detectable at day 30.22 Information from the product sourcea indicates that the matrix slowly dissolves over 4 to 5 weeks. The cisplatin-impregnated beads have been used safely and effectively to treat cutaneous tumors in horses17,18 and soft tissue sarcomas in dogs.21 However, to the authors' knowledge, there are no published studies evaluating the use of carboplatin-impregnated biodegradable beads in dogs.

Compounded drugs are not approved by the US FDA, and the agency does not verify the safety or efficacy of these drugs.23 Although, in the United States, state boards of pharmacy oversee state-licensed pharmacies to ensure that drugs are compounded in accordance with the conditions of section 503A of the Federal Food, Drug, and Cosmetic Act and current compliance policy guides, no direct FDA finding of manufacturing quality is required before compounded drugs are marketed. A recent study24 found that the potency of lomustine from 5 different compounding pharmacies ranged from 50% to 115% of the labeled concentration, with only 1 sample within 10% of the labeled concentration, suggesting that there can be substantial variability in some compounded products.

The purpose of the study reported here was to prospectively evaluate the platinum content in carboplatin-impregnated beads obtained from a single accredited compounding pharmacy and to retrospectively evaluate tolerability and outcome data for dogs treated by intralesional placement of the beads following surgical excision of subcutaneous sarcomas. We hypothesized that each bead would contain approximately 2.4 mg of platinum (equivalent to the labeled content of 4.6 mg of carboplatin) and that the results of retrospective analysis would reveal that the beads were well tolerated and provided local control of soft tissue sarcomas similar to that previously reported for dogs treated with cisplatin-impregnated beads.21

Materials and Methods

In vitro experiments

Biodegradable carboplatin beads—Three-millmeterdiameter calcium sulfate and dextran sulfate matrix carboplatin-impregnated beadsb were prepared by an accredited compounding pharmacy.c The labeled amount of carboplatin per bead was 4.6 mg. The beads were shipped in 3 sterile vials, each containing 3 beads; each vial was from a different lot. Beads were stored at room temperature, and content analysis was performed prior to the labeled expiration dates.

Evaluation of platinum content—Each bead (n = 9) was analyzed in 1 run for total platinum content by means of a qualified atomic absorption method. The study samples were quantitated by comparison with calibration standards prepared from a platinum reference standardd (1,000 μg/mL of stock) in a 0.14% HNO3 diluent solution with 0.1% nonionic surfactant added.e Quality control samples were prepared similarly. The calibration range for total platinum concentration was 25 to 800 ng/mL, and quality control samples were prepared at 75-, 250-, and 600-ng/mL concentrations. The beads were homogenized in diluent with a blenderf and then adjusted to a concentration of 1.0 mg of bead/mL. Samples were further diluted to 1:100,000 in the same diluent to bring the platinum concentrations into the calibration curve range. A 20-μL aliquot of sample was then injected into a graphite furnace atomic absorption spectrophotometerg for analysis with associated software. Transversely heated graphite atomizer tubes with end capsh were used for the analysis. The graphite furnace program consisted of a 2-stage drying step at 130° and 150°C; pyrolysis at 1,400°C; atomization at 2,200°C; and cleanout at 2,500°C with a read time of 5 seconds and baseline offset correction set at 2 seconds. The baseline offset correction set the baseline just prior to atomization. The needle wash consisted of 1.4% nitric acid solution. Analyte was detected with a platinum hollow cathode lamp (primary wavelength, 265.9 nm; low slit width, 0.7 nm; lamp current, 30 mA). Calibration curves were determined by plotting analyte area response versus known concentrations (ng/mL) and performing non-weighted linear regression. Concentrations were back-calculated with the formula × = (y – b)/m, where × is the back-calculated concentration, y is the analyte area response, b is the y-intercept, and m is the slope. Calibrator and quality control acceptance criteria required all acceptable concentrations to have accuracy deviations of ≤ 15% from the nominal concentration with relative SDs of ≤ 15%, except at the lower limit of quantitation, which was allowed up to a 20% deviation for both variables.

Retrospective investigation of clinical cases

Case selection—Client-owned dogs with histologically confirmed subcutaneous sarcomas that were evaluated at Oradell Animal Hospital between February 6, 2011, and October 23, 2014, were considered for inclusion in the retrospective evaluation of treatment tolerability and outcomes. Dogs with visible disease at the primary tumor site or a primary tumor that was incompletely excised < 1 month prior to this examination were eligible for the study. Dogs with tumors that had recurred locally after previous treatment by surgery, radiation therapy, chemotherapy, or a combination of these approaches were allowed in the study; the measurable local tumor burden had to be amenable to surgery such that all gross evidence of the tumor could be removed, but curative local control was deemed unlikely with surgery alone.

Results of a baseline (within 1 month prior to surgery) CBC, serum biochemical analysis, and urinalysis as well as thoracic radiography were required for all study dogs. Dogs with concurrent diseases were enrolled, provided that each dog had been deemed healthy enough to safely undergo anesthesia and surgery and that its estimated life expectancy was > 1 month. Similarly, dogs with evidence of early pulmonary metastasis were included if it was documented that the clinician believed the primary tumor was likely to affect the patient's quality of life before the metastatic disease would. Dogs that had adjuvant chemotherapy were included, provided that the treatment was not started until any postoperative adverse events attributable to the local treatment had completely resolved. All dog owners had provided informed consent prior to surgery and bead implantation, and each patient was treated according to the contemporary standard of care.

Medical records review—Signalment; type, MI, and location of the tumor; measurement of the tumor at its greatest diameter; and any prior treatments were recorded from the medical records of each dog. Surgical information collected included the date of surgery and the number of carboplatin-impregnated beads placed. Histopathologic data, including margin assessment, and any adverse events reported by the owners at the initial postoperative examination (10 to 14 days after surgery) were recorded. Adverse events were classified by use of previously described criteria as mild (fluctuant swelling without drainage or tissue necrosis), moderate (swelling with heat, erythema, drainage, and wound breakdown without tissue necrosis), or severe (drainage with tissue necrosis).12,13,21,25 Although the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events26 was created to define and grade adverse events resulting from systemic chemotherapy treatments rather than from surgery or local chemotherapy, the authors also graded adverse events according to the scheme for injection site reactions listed under administration site conditions in that document as follows: grade 1 = tenderness with or without warmth or erythema; grade 2 = edema with signs of pain; grade 3 = ulceration, necrosis, or other severe tissue damage with surgical intervention indicated; and grade 4 = life-threatening consequences requiring urgent intervention. Follow-up data from subsequent recheck examinations, including any delayed adverse events associated with surgery and any evidence of local tumor recurrence, were recorded. When necessary, referring veterinarians and dog owners were contacted by telephone for updates.

Surgery, postoperative care, and follow-up—All surgeries were performed by 1 of 3 board-certified veterinary surgeons (JMM, AAF, or CAC). Each tumor or surgical scar was excised with appropriate margins laterally (≥ 3 cm) and deep (≥ 1 fascial plane) when anatomically possible. After adjusting for wound closure, carboplatin-impregnated beads were placed approximately 1.5 cm apart along an imaginary 2-D grid across the entirety of the wound. When practical, each bead was placed under a fascial layer or through a small stab incision into a muscle belly as applicable. Sutures were placed to close fascia over the beads, then routine closure of subcutaneous tissue and skin was performed. Placement of surgical drains was not performed. All tissue removed was submitted for histologic evaluation and margin assessment.

All patients were hospitalized overnight and received perioperative analgesics and antimicrobials; however, the type and duration of these treatments were not standardized. Warm compresses over the surgical site were not allowed because the resulting increase in blood circulation might have altered distribution of the chemotherapeutic agent. An Elizabethan collar was placed as necessary to prevent dogs from licking the incision site.

At the time of each dog's discharge from the hospital, owners were asked to monitor the patient for any evidence of pain or discomfort as well as swelling, discharge, or redness of the surgical site and to report any abnormalities observed. They also were provided written and verbal instructions cautioning that any discharge could potentially contain the chemotherapeutic drug. Owners were advised to wear latex or nitrile gloves if they needed to clean the incision, handle any contaminated bedding, or clean any contaminated environmental surfaces.

All dogs returned for postoperative evaluation 10 to 14 days after surgery. Skin sutures or staples were removed at this follow-up visit, and the incision site was examined for any evidence of abnormalities in the healing process. Long-term follow-up recommendations included recheck examinations every 1 to 2 months for the first year, then every 2 to 3 months thereafter, but compliance with the recheck recommendations varied.

Statistical analysis

Summary statistics were used to describe the total platinum content in the compounded beads within and among lots. A Shapiro-Wilk test was used to assess data distribution. Because atomic absorption spectrophotometry only measured platinum content, the corresponding amount of carboplatin was calculated by multiplying the amount of platinum by 1.903. This conversion factor was derived by dividing the molecular weight of carboplatin (C6H12N2O4Pt = 371.248) by the atomic weight of platinum (195.084). A 1-sample t test was used to compare the mean calculated carboplatin content with the labeled carboplatin content of 4.6 mg/bead. Differences among the 3 lots were evaluated by means of a 1-way ANOVA, and when significant differences were identified, a post hoc Tukey test was used to compare results among lots.

For each dog, the carboplatin dose administered was first calculated on the basis of the labeled amount of 4.6 mg of carboplatin/bead. The absolute dose, the dose calculated according to the dog's BSA, and the fractional dose relative to a standard IV carboplatin dose (300 mg/m2 of BSA) were calculated. This analysis was then repeated with the carboplatin dose calculated on the basis of the mean carboplatin content per bead derived from the spectrophotometric analysis.

Dogs were dichotomized according to the presence or absence of any adverse events after surgery. Tumor location (trunk [including the neck] vs limbs), MI (< 20 mitoses/10 hpf vs ≥ 20 mitoses/10 hpf, a cutoff that is significant for metastasis and survival2), and histologic margins (neoplastic cells present or absent at margins evaluated) were evaluated for significant associations with any adverse events by means of a Fisher exact test. The tumor diameter in its greatest dimension, absolute number of carboplatin-impregnated beads placed, number of beads relative to the greatest tumor diameter, and number of beads relative to the dog's BSA were evaluated for significant associations with any adverse events by use of a 2-sided Wilcoxon rank sum test.

Long-term prognosis was not a primary aim of this study; however, local DFI was evaluated with the Kaplan-Meier product limit method. Local DFI was defined as the time from surgery and carboplatin-impregnated bead implantation until local recurrence at the site of the primary tumor was identified by physical examination (palpation) or cytologic or histologic assessment. Dogs were censored if they had no evidence of disease at the primary tumor site at the time of last follow-up or at the time of death. A log-rank test was used to compare Kaplan-Meier curves when patients were dichotomized on the basis of tumor location (trunk vs limbs), MI (< 20 mitoses/10 hpf vs ≥ 20 mitoses/10 hpf), histologic margins (neoplastic cells present or absent at margins evaluated), any previous local treatment for the sarcoma being evaluated (yes or no), other postoperative chemotherapy treatments (yes or no), longest tumor diameter (whether above or below the median value), absolute number of carboplatin-impregnated beads placed (above or below the median), relative platinum dose (mg/m2 of BSA; above or below the median), and number of beads placed per centimeter of the greatest tumor diameter (above or below the median).

All statistical calculations were performed with a computer software program.i For all tests, values of P < 0.05 were considered significant.

Results

In vitro experiments

All carboplatin-impregnated beads purchased for in vitro testing were analyzed in 1 analytical run. The quality controls had overall mean ± SD accuracy of 104 ± 2.31% and precision of 4.23 ± 2.29%. The correlation coefficient for the calibration curve was ≥ 0.9999. The results for the platinum bead analysis were summarized (Table 1). The overall mean ± SD platinum content per bead was 5.38 ± 0.97 mg, and the corresponding mean calculated amount of carboplatin per bead was 10.24 ± 1.84 mg. The calculated mean carboplatin content per bead was significantly (P < 0.001) greater than the labeled content of 4.6 mg/bead. The absolute amount of platinum per bead differed significantly (P = 0.004) among lots, with the beads in lot 2 containing greater amounts than those in lots 1 and 3 (P < 0.05 for both post hoc comparisons). Similarly, there were significant (P = 0.019) differences in bead weight among lots, with the beads in lot 2 weighing significantly (P < 0.05 for both post hoc comparisons) more than those in lots 1 and 3. The concentration of platinum per milligram of bead weight was not significantly (P = 0.97) different among lots.

Table 1—

Results of analysis for platinum content by atomic absorption spectrophotometry in 9 compounded carboplatin-impregnated beads of 3 lots obtained from 1 source.

Lot no.Bead weight (mg)Total platinum content per bead (mg)Calculated carboplatin content per bead (mg)*Total platinum content per mg of bead weight (mg/mg)
Mean ± SDRangeMean ± SDRangeMean ± SDRangeMean ± SDRange 
120.1 ± 3.717.7–24.44.81 ± 0.714.29–5.619.15 ± 1.348.16–10.680.24 ± 0.010.23–0.24
227.4 ± 1.126.5–28.76.57 ± 0.146.41–6.6612.50 ± 0.2612.20–12.680.24 ± 0.010.22–0.25
320.1 ± 2.118.6–22.54.76 ± 0.254.58–5.049.06 ± 0.478.72–9.600.24 ± 0.010.22–0.25
All22.6 ± 4.2617.7–28.75.38 ± 0.974.29–6.6610.24 ± 1.848.16–12.680.24 ± 0.010.22–0.25

Beads were homogenized in 0.14% HNO3 diluent solution with 0.1% nonionic surfactant added, and the samples were adjusted to bring the nominal platinum concentrations into the range of the calibration curve (25.0 to 800.0 ng/mL).

The amount of carboplatin was calculated by multiplying the measured amount of platinum by 1.903 (a conversion factor derived by dividing the molecular weight of carboplatin [371.248] by the atomic weight of platinum [195.084]).

Retrospective investigation of clinical cases

Twenty-nine dogs were included in the retrospective analysis. The breeds most commonly represented were Golden Retriever (n = 5), Labrador Retriever (3), and Wheaten Terrier (2), with 1 each of the following breeds: Beagle, Bullmastiff, Cairn Terrier, Collie, German Shorthaired Pointer, Maltese, Pomeranian, Rhodesian Ridgeback, Rottweiler, Shetland Sheepdog, Vizsla, and West Highland White Terrier. Seven dogs were mixed breed. Ten of 29 (34%) dogs were spayed females, and 19 (66%) were neutered males. The median age was 10.8 years (range, 1.3 to 14.9 years), and median weight was 27.5 kg (60.5 lb; range, 7.7 to 59.3 kg [16.9 to 130.5 lb]).

Data for 29 tumors were included in the study. One dog had 3 noncontiguous anaplastic sarcomas that were treated with surgery and carboplatin-impregnated bead implantation at various times. Another dog underwent a second surgery and bead implantation for the same hemangiopericytoma after it recurred locally. For both dogs, only the first surgery and bead implantation were included in the study. Overall, there was no prior treatment for 17 of 29 (59%) tumors. For the remaining 12 (41%) tumors, prior treatments included surgery alone (n = 6) or surgery with ≥ 1 of the following: radiation therapy (4), IV chemotherapeutic treatment (1), and metronomic chemotherapy (oral treatment; 1).

For 27 of 29 (93%) tumors, there was gross disease present immediately prior to surgery and carboplatin-impregnated bead implantation, and the median maximum tumor diameter was 4.5 cm (range, 2.0 to 20.0 cm). For the other 2 (7%) tumors, there was only residual microscopic disease left behind from prior incomplete excision. The length of the surgical scars after incomplete excision of these tumors was 2.5 cm and 6.0 cm. The intervals between the first surgery and the second surgery, which included bead implantation, were 14 and 21 days, respectively.

The histologic diagnoses for sarcomas were unclassified spindle cell tumor or soft tissue sarcoma (n = 8), hemangiopericytoma (7), peripheral nerve sheath tumor (5), hemangiosarcoma (3), anaplastic sarcoma (2), liposarcoma (1), malignant fibrous histiocytoma (1), myxofibrosarcoma (1), and soft tissue recurrence of a rib osteosarcoma (1). Sixteen of 29 (55%) tumors were axial, and 13 (45%) were located on limbs (7 on pelvic limbs and 6 on thoracic limbs). Twenty-four (83%) tumors had an MI < 20 mitoses/10 hpf, and 5 (17%) had an MI ≥ 20 mitoses/10 hpf (median for these 5 tumors, 31 mitoses/10 hpf; range, 21 to 42 mitoses/10 hpf). The tumors with an MI ≥ 20 mitoses/10 hpf included anaplastic sarcoma (n = 1), hemangiosarcoma (1), liposarcoma (1), malignant fibrous histiocytoma (1), and unclassified soft tissue sarcoma (1). Evaluation of the 29 histopathology reports for tissues collected during surgeries in which carboplatin-impregnated beads were implanted revealed that neoplastic cells were identified at the surgical margins of 15 (52%) tumors and were not seen at the surgical margins in the remaining 14 (48%) tumors. Of the latter 14 tumors, 9 had very narrow margins (< 1 to 2 mm) and thus complete excision could not be confirmed. Five tumors had margins ≥ 1 cm, although 2 of these tumors had recurred following previous local treatments (surgery with or without radiation therapy), making it difficult to confirm complete excision.

The median number of beads implanted for each tumor was 9 (range, 3 to 20 beads). When the number of beads was divided by tumor diameter in the greatest dimension, the median value was 1.56 beads/cm (range, 0.45 to 3.60 beads/cm). On the basis of the manufacturer's labeled content of 4.6 mg of carboplatin/bead, the median absolute carboplatin dose was 41.4 mg (range, 13.8 to 92 mg). Calculated relative to BSA, the median dose on the basis of labeled content was 39.0 mg/m2 (range, 11.0 to 104.9 mg/m2). Compared with a standard 300-mg/m2 IV dose of carboplatin, the median relative dose was 13.0% (range, 3.7% to 35.0%).

Results of the vitro experiments indicated that the platinum content in the beads was variable. However, if the beads used in those experiments had similar characteristics to those used in the patients of this study, the extrapolated median absolute dose of carboplatin would be 92.2 mg (range, 30.7 to 204.8 mg) on the basis of the calculated mean carboplatin content of 10.24 mg/bead. Calculated relative to BSA, the extrapolated median dose would be 86.6 mg/m2 (range, 24.5 to 233.5 mg/m2), and compared with a standard 300-mg/m2 IV dose of carboplatin, the median relative dose would be 28.9% (range, 8.2% to 77.8%).

Adverse events were identified after 11 of 29 (38%) surgeries. Adverse events were considered mild or grade 1 following surgery in 8 dogs; these included seroma (n = 6), mild swelling (1), and formation of annular crusting lesions adjacent to the incision (1). Adverse events were considered moderate or grade 2 for the remaining 3 dogs, with mild erythema (n = 1), mild edema with serosanguineous discharge (1), and purulent bloody discharge (1) observed. All adverse events resolved completely ≤ 1 month after surgery. Additional supportive care provided at the discretion of the attending clinician included oral administration of NSAIDs, antimicrobial treatments, use of Elizabethan collars, and bandage placement, alone or in combination. None of the adverse events required a second surgery or removal of the implants. No beads were visible or found to be extruding from the wound site during the healing process. No signs of systemic toxicosis (eg, anorexia, vomiting, or diarrhea) were reported. None of the variables evaluated were significantly associated with development of postoperative adverse events.

Twelve of 29 (41%) dogs had tumors treated by adjuvant chemotherapy after the surgery site was completely healed. Three dogs (with hemangiosarcoma [n = 2] or osteosarcoma [1]) received IV doxorubicin treatment. The remaining 9 were treated with various metronomic chemotherapy protocols, including single-agent chlorambucil (n = 3), single-agent cyclophosphamide (2), cyclophosphamide with piroxicam (2), cyclophosphamide with meloxicam (1), and single-agent toceranib phosphate (1).

At the time of analysis, 9 of 29 (31%) tumors had recurred locally and 20 (69%) had not. For the 9 tumors that did recur, median time to recurrence was 84 days (range, 54 to 759 days). For the 20 tumors that did not recur, median duration of follow-up was 532 days (range, 52 to 1,572 days). An overall median local DFI was not reached (Figure 1). The 1-, 2-, and 3-year local disease-free rates determined by Kaplan-Meier analysis were 70%, 70%, and 58%, respectively.

Figure 1—
Figure 1—

Kaplan-Meier curve showing the local DFI for subcutaneous sarcomas in 29 dogs treated with marginal excision and intraoperative placement of biodegradable carboplatin-impregnated beads. Median DFI was not reached. Local disease-free rates at 1, 2, and 3 years were 70%, 70%, and 58%, respectively. Vertical lines represent censored data.

Citation: Journal of the American Veterinary Medical Association 252, 4; 10.2460/javma.252.4.448

Tumors with an MI < 20 mitoses/10 hpf had a significantly (P = 0.019) longer local DFI (median DFI not reached) than did those with an MI ≥ 20 mitoses/10 hpf (median DFI, 102 days). The local DFI was significantly (P = 0. 033) longer for tumors that did not have neoplastic cells identified at the surgical margins (median DFI not reached), compared with tumors that did have neoplastic cells identified at the surgical margins (median DFI, 759 days). Finally, the local DFI was significantly (P = 0.014) longer for dogs that were not treated with postoperative adjuvant chemotherapy (median DFI not reached), compared with dogs that did receive postoperative adjuvant chemotherapy (median DFI, 102 days). The other variables evaluated were not significantly associated with DFI.

Discussion

The calculated carboplatin content in biodegradable carboplatin-impregnated beads in the present study (mean ± SD, 10.24 ± 1.84 mg/bead) was significantly higher than the labeled content of 4.6 mg/bead. Atomic absorption spectrophotometry measures total platinum, rather than specifically intact carboplatin molecules. However, this technique is commonly used in studies evaluating carboplatin pharmacokinetics in a variety of species.27–31 High-performance liquid chromatography would be needed to measure unchanged carboplatin, but when these 2 techniques were compared, the results were considered interchangeable.31 Additionally, beads manufactured from the same matrix, when impregnated with cisplatin, were shown to be stable for up to 1 year,18 and similar stability has been described in the manufacturer's product information sheet32 for the carboplatin-impregnated beads used in the present study. All beads used in the present study were evaluated before their labeled expiration date. Therefore, it is unlikely that a clinically relevant amount of carboplatin would have degraded in the beads. Because the matrix is biodegradable, there was also no reason to expect incomplete elution of the carboplatin from the beads.

We also identified statistically significant differences among the 3 lots tested for bead weight and absolute platinum content. However, the amount of platinum per milligram of bead weight was consistent. On the basis of these data, it is reasonable to consider that the carboplatin was mixed with the matrix at a uniform ratio, but the amount of matrix-carboplatin mixture that was extruded into the individual beads varied substantially.

Drugs approved by the FDA undergo rigorous testing with regard to drug content, stability, and accuracy relative to the labeled contents (usually within 10% to 20%) between and within lots.23 Similar mechanisms do not exist for compounded products. Neither the cisplatin-impregnated beads used in previous studies18,21 nor the carboplatin-impregnated beads used in the present study are approved by the FDA for use in animals, although the cisplatin-impregnated beads have an approved Investigational New Animal Drug Application.33 The matrix has been licensed to a single veterinary compounding pharmacy that manufactures the beads in accordance with the United States Pharmacopeia Convention guidelines for pharmaceutical compounding of sterile preparations.j Prior to sale and distribution, each completed batch is tested for sterility, but it is not tested for drug concentration.j

The absolute amount of platinum in the beads tested in the in vitro portion of our study was variable, and the calculated carboplatin content was considerably higher than the labeled content. Only a small number of lots were evaluated, and they were not necessarily the same lots used in the dogs of this study. However, if the actual platinum content in the beads used was higher than the labeled amount, their use still was well tolerated. Local adverse events (wound complications) were observed following surgery and bead implantation for 11 of 29 (38%) dogs. The complications were considered mild in 8 cases and moderate in 3; all adverse events resolved completely ≤ 1 month after surgery, and none of the dogs required additional follow-up surgery as a result of adverse events. A recently published study21 that investigated cisplatin-impregnated beads of the same biodegradable matrix reported local toxic effects in 24 of 51 (47%) dogs. In that study,21 toxic effects were considered mild in 12 dogs, moderate in 10, and severe in 2.

When open-cell polylactic acid sponges containing cisplatin were used in combination with marginal surgery to treat soft tissue sarcomas in dogs in a previous study,13 wound complications were observed in 15 of 32 (47%) dogs, and surgical removal of the implant was required for 9 (28%) dogs. When another polymer-based cisplatin-containing implant was used for this purpose, wound complications were observed in 16 of 19 dogs, and surgical removal of the implant was deemed necessary for all 16 dogs with complications.12 The reasons for the apparent differences in wound complication rates or severities between the described studies and the present study could not be determined; however, the drug delivery system, the specific chemotherapeutic drug used, some aspect of surgical technique, the amount of chemotherapeutic agent used, and other patient-related factors all could have contributed.

Adverse systemic effects were not observed in any of the dogs of the present study; however, CBCs were not performed to assess neutrophil and platelet counts during treatment of these dogs, and this was not ideal. Systemic toxicosis was rarely reported in other studies12,13,21 in which local chemotherapy with platinum agents was used. Additionally, the chemotherapy dose on a mg/m2 of BSA basis delivered to the patients in the present study was well below the amount expected to cause clinically relevant myelosuppression. In a phase I evaluation34 of carboplatin administered IV to dogs, clinically relevant myelosuppression was not identified in any dogs treated at doses ranging from 100 to 200 mg/m2. On the basis of the labeled dose of 4.6 mg carboplatin/bead, the highest dose administered to any dog in this study was 104.9 mg/m2. Extrapolating from the mean value determined in our in vitro study, if the carboplatin content in the beads used was 10.24 mg/bead, the highest dose would have been 232.5 mg/m2. Thus, we believe that clinically relevant myelosuppression was unlikely, but this cannot be definitively stated. One of 62 dogs in a study21 that evaluated the use of cisplatin-impregnated beads implanted SC for treatment of soft tissue sarcomas had presumptive neutropenic sepsis with vomiting and diarrhea, which developed 14 days after bead implantation. On the basis of labeled drug content, that patient had received a total cisplatin dose of 51.69 mg/m2, whereas the median total cisplatin dose was 15.5 mg/m2 for all patients in the study.21 It is also worth noting that the authors of the present study have detected grade 3 neutropenia (610 neutrophils/μL; reference range, 1,480 to 10,290 neutrophils/μL) in a 4.0-kg (8.8-lb) cat that had 17 carboplatin-impregnated beads implanted for treatment of a large vaccine-associated sarcoma (unpublished clinical data). Therefore, we recommend that caution be used in small patients with large tumors where the implanted dose might approach the amount of drug delivered in a standard IV dose of carboplatin.

Two of 29 (7%) dogs in the present study developed small amounts of discharge from the surgical site. We were not able to analyze the fluids to determine whether they contained detectable concentrations of the chemotherapeutic agent, and previous studies12,13,21 evaluating intraoperative placement of platinum drug-containing implants did not specifically address leakage of chemotherapeutic agents from the surgical wound. When aqueous carboplatin was injected intratumorally at 6- to 8-mm intervals in cats with squamous cell carcinoma of the nasal plane, approximately 10.9% and 3.3% of the dose leaked from the injection site when the drug was used alone or mixed with sterile sesame oil, respectively.16 The method of drug delivery can thus be expected to influence environmental contamination, and without such safety studies for the beads under investigation, it must be assumed that any fluid leaked from the surgical site (eg, postoperative bleeding or other discharge) potentially contains chemotherapeutic agent. In vitro, the majority of platinum elutes ≤ 72 hours after placement in PBS solution, but some platinum elution still occurs at day 30.22 In the present study, the number of dogs with confirmed discharge from the surgical site was low, but the actual incidence could have been higher. Some owners might not have recognized very small amounts of discharge, and dogs allowed to lick the incision site could potentially have ingested discharged fluid without it being recognized.

Environmental contamination and unintended exposure to people and animals are risks associated with chemotherapeutic treatment of pets. In 1 study,35 dogs that received carboplatin IV were shown to have detectable concentrations of platinum primarily in urine, but also in feces and saliva, for up to 21 days after treatment. However, the use of a local implant provides an additional mechanism and source for potential contamination, and the aspects of human and home environment safety must be carefully considered and discussed with the owner before such therapy is recommended. The possibility of exposure to chemotherapeutic agents through local discharge was discussed with all of our clients prior to implant surgery, and instructions for wound care and environmental cleaning were provided verbally and in writing. We also hospitalized all patients for 1 night to ensure there were not any immediate complications at the surgical site. It would be reasonable to consider hospitalizing patients for longer periods to help ensure there is no environmental contamination from the surgical wound. Bandages could also be used, depending on the anatomic location of the surgical wound, to help minimize the risk of exposure to chemotherapeutic drugs.18

The use of intraoperatively placed implants also has theoretical advantages over other forms of chemotherapeutic treatment. Intralesional administration helps maximize drug concentrations locally, but as in the present study, the total dose delivered to the patient is typically lower than the standard IV dose. Additionally, intraoperative placement of the implant allows the chemotherapeutic agent to be distributed more precisely within the selected treatment field, particularly for large or deep tumors.

Outcome and local DFI data from the present study must be interpreted cautiously. Our primary intent was to evaluate tolerability of the treatment, and therefore, tumor type was not standardized in this study. Dogs with subcutaneous or intramuscular hemangiosarcoma (n = 3) or soft tissue recurrence of a primary rib osteosarcoma (1) were included in the study; these tumors have more aggressive biological characteristics36–39 than do some other types of subcutaneous soft tissue sarcomas such as fibrosarcoma, hemangiopericytoma, and peripheral nerve sheath tumors.1–7 Additionally, even among the remaining 25 subcutaneous soft tissue sarcomas, 5 (20%) had an MI ≥ 20/hpf, 11 (44%) had recurred after previous treatments, and 9 (36%) were treated with adjuvant oral metronomic chemotherapy. The median local DFI was shorter for all tumors that received adjuvant treatments than for those that did not, and this likely reflected the use of such treatments for tumors expected to have more aggressive biological characteristics.

In studies2,6,7 evaluating dogs with subcutaneous soft tissue sarcomas treated by surgery alone, reported local recurrence rates for incompletely excised tumors range from 4 of 24 (17%) to 55 of 55 (100%). This wide range likely reflects differences in surgical technique, duration of follow-up, and heterogeneity of tumor characteristics. This variability emphasizes the limitations of making comparisons across studies. Prospective, randomized trials are needed to better define the role of local chemotherapy with carboplatin-containing implants for incompletely excised soft tissue sarcomas in dogs and to allow accurate comparisons with other adjuvant treatments such as radiation therapy and low-dose oral antiangiogenic (metronomic) chemotherapy with respect to efficacy, toxic effects, and cost-benefit investigations.7–9 However, our results suggested that better manufacturing quality control is needed for any further clinical investigation to have meaningful results, and that caution must be exercised when using this product. Studies assessing environmental contamination and owner safety are needed as well.

Acknowledgments

The authors report no external sources of funding or conflicts of interest.

Presented in poster form at the 35th Annual Veterinary Cancer Society Forum, Las Vegas, October 2012.

ABBREVIATIONS

BSA

Body surface area

DFI

Disease-free interval

MI

Mitotic index

Footnotes

a.

Tatiana Bizikova, Royer Biomedical Inc, Frederick, Md: Personal communication, 2015.

b.

Matrix III, Royer Animal Health, Frederick, Md.

c.

Wedgewood Pharmacy, Swedesboro, NJ.

d.

Spex Certiprep, Metuchen, NJ.

e.

Triton X-100, Sigma-Aldrich Corp, St Louis, Mo.

f.

Bullet Blender, Storm 24, NextAdvance, Averill Park, NY.

g.

AAnalyst 600 atomic absorption spectrophotometer with WinLab 32 software, Perkin-Elmer, Branford, Conn.

h.

THGA graphite tubes, catalog No. B3000653, Perkin-Elmer, Branford, Conn.

i.

GraphPad Prism, version 5.0, GraphPad Software Inc, San Diego, Calif.

j.

Staff Pharmacist, Wedgewood Pharmacy, Swedesboro, NJ: Personal communication, 2015.

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