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Keywords:

  • Chemotherapy;
  • Lymphoma;
  • MDR1;
  • P-glycoprotein;
  • Pharmacogenetics

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background: Dogs that harbor the naturally occurring ABCB1-1Δ polymorphism experience increased susceptibility to avermectin-induced neurological toxicosis as a result of deficient P-glycoprotein function. Whether or not the ABCB1-1Δ polymorphism affects susceptibility to toxicity of other P-glycoprotein substrate drugs has not been studied.

Hypothesis: Dogs that possess the ABCB1-1Δ mutation are more likely to develop hematologic toxicity associated with vincristine than ABCB1 wild-type dogs.

Animals: Thirty-four dogs diagnosed with lymphoma were included in this study.

Methods: Cheek swab samples were obtained from dogs diagnosed with lymphoma that were to be treated with vincristine. DNA was extracted from cheek swabs and the ABCB1 genotype was determined. Hematologic adverse drug reactions were recorded for each dog and graded according to the Veterinary Comparative Oncology Group's criteria for adverse event reporting (Consensus Document). In order to avoid possible bias, ABCB1 genotype results for a particular patient were not disclosed to oncologists until an initial adverse event report had been submitted.

Results: Dogs heterozygous or homozygous for the ABCB1-1Δ mutation were significantly more likely to develop hematologic toxicity, specifically neutropenia (P= .0005) and thrombocytopenia (P= .0001), after treatment with vincristine than ABCB1 wild-type dogs.

Conclusions and Clinical Implications: At currently recommended dosages (0.5–0.7 mg/M2), vincristine is likely to cause hematologic toxicity in dogs with the ABCB1-1Δ mutation, resulting in treatment delays and unacceptable morbidity and mortality. Assessing the ABCB1-1Δ genotype before vincristine administration and decreasing the dosage may prevent toxicity and treatment delays resulting from neutropenia or thrombocytopenia.

Vincristine, a vinca alkaloid, exerts cytotoxic effects by binding to microtubular proteins and thereby interfering with mitosis and arresting susceptible cells in metaphase. In dogs with cancer, vincristine is used most commonly in combination chemotherapy protocols to treat lymphoma. Adverse effects of vincristine include peripheral neuropathy, gastrointestinal toxicity, and myelosuppression.1–3 At standard dosages (0.5–0.7 mg/M2)1,3,4 vincristine is not usually myelosuppressive in dogs,1,3 but hematologic toxicity, primarily neutropenia, can occur in some patients and may result in treatment delays. Because treatment delays may decrease the likelihood of achieving remission, owners' compliance to continue treatment, or shortening remission duration, avoidance of vincristine-induced hematologic toxicity is desired. Furthermore, complications such as sepsis are associated with neutropenia and can increase patient morbidity and mortality, providing further incentive for avoiding hematologic toxicity. Currently, there are no parameters that can be used to predict which dogs are likely to experience hematologic toxicity from vincristine. One recent study of canine lymphoma reported that 15.6% of dogs required vincristine dose reduction because of development of toxicity.4 The ability to identify dogs that will develop hematologic toxicity before vincristine treatment would be clinically useful to provide maximum chemotherapeutic efficacy while minimizing toxicity.

Pharmacogenetics is the study of how an individual's genetic make-up determines its response to drugs. In humans, interindividual variation of drug toxicity and efficacy mainly is determined by genetic polymorphisms in drug metabolizing enzymes, receptors, or drug transporters.5 For example, the anticancer drug irinotecan can cause severe gastrointestinal and hematologic toxicity in patients with certain polymorphisms in the UDP-glucuronyl transferase gene.6 These polymorphisms result in defective UDP-glucuronyl transferase activity and ultimately decreased clearance of active drug. In dogs, vincristine is eliminated primarily by biliary excretion of parent drug with some urinary excretion of parent drug and metabolites.2,7 The enzyme family responsible for metabolizing vincristine in dogs has not been identified. Biliary excretion of vincristine in other species is highly dependent on the drug transporter P-glycoprotein, which is present on biliary canalicular cells and renal tubular epithelial cells.8 Because P-glycoprotein function is deficient in Herding breed dogs with the ABCB1-1Δ (formerly known as MDR1) mutation,9 we postulated that these dogs may be more susceptible to vincristine toxicity. A recent case report described hematologic and gastrointestinal toxicity in a dog with the ABCB1-1Δ mutation at normal and decreased doses of vincristine, but the patient tolerated full doses of cyclophosphamide,10 a chemotherapeutic drug that does not incur resistance via P-glycoprotein. The purpose of this prospective study was to assess the role of the ABCB1-1Δ mutation in the occurrence of hematologic toxicity in dogs receiving vincristine.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Study Population and Procedure

This study consisted of 34 dogs undergoing chemotherapy for lymphoma using any vincristine-containing protocol. Buccal cells were obtained from each dog and submitted for ABCB1 genotyping, and the dog's breed and dosage of vincristine was recorded. Initial accessions included all dog breeds to ensure an adequate population of ABCB1-1Δ wild-type dogs, but because the ABCB1-1Δ mutation has been identified only in herding breed dogs, later accessions were limited to herding breeds. Dogs were treated based on the institutions' standard treatment practices for canine lymphoma, with hematologic adverse reactions attributed to vincristine administration graded according to the Veterinary Comparative Oncology Group's (VCOG)11 criteria for adverse event reporting with 1 modification (Table 1). The modification was simply assigning a grade of 0, for statistical purposes, in categories in which the patient did not experience an adverse event. Dogs that had received a chemotherapeutic drug, with the exception of a corticosteroid, concurrently with vincristine were excluded from study, with 1 exception. If a dog had received l-asparaginase concurrently with vincristine and the dog did not experience an adverse event, that patient was included in the study. A CBC obtained between 5 and 15 days after vincristine treatment was used to determine vincristine-associated hematologic toxicity. Abnormalities in neutrophils and thrombocytes were included in statistical analysis. Data for hemoglobin and PCV were not analyzed in this study because it is unlikely that these 2 parameters could be directly linked to vincristine within the 5–15 day period established to observe for vincristine-induced hematologic toxicity. Because the average lifespan of a red blood cell in dogs is roughly 100–120 days, it is unlikely that PCV and hemoglobin concentrations would be affected within that time period as a direct result of drug-induced myelosuppression.

Table 1.   Criteria for grading severity of adverse events related to blood and bone marrow modified from a consensus document from the Veterinary Cooperative Oncology Group.11
 Blood/Bone Marrow
Adverse EventGrade
012345
  1. LLN, lower limit of normal.

NeutropeniaWithin normal limits1,500/μL to <LLN1,000–1,499/μL500–999/μL<500/μL
ThrombocytopeniaWithin normal limits100,000/μL to <LLN50,000–99,999/μL25,000–49,999/μL<25,000/μL

ABCB1 genotyping was performed according to previously published methods.12 ABCB1 genotypes were designated in the following manner: dogs with 2 ABCB1 wild-type alleles = ABCB1 wt/wt, dogs with 1 ABCB1 wild-type allele and 1 ABCB1-1Δ allele = ABCB1 wt/mut, and dogs with 2 ABCB1-1Δ allele = ABCB1 mut/mut. In order to avoid possible bias, ABCB1 genotype results were not made available to oncologists until after the adverse event form had been submitted.

Statistical Analysis

A score of 0 was assigned if an adverse event did not occur, and a score of 1 was assigned if an adverse event did occur (score of ≥1 on the VCOG common terminology criteria for adverse events after chemotherapy) (Table 1). Separate χ2 analyses were performed to assess the overall association between the occurrence of neutropenia or thrombocytopenia and the patient's genotype (ABCB1 wt/wt; ABCB1 wt/mut, or ABCB1 mut/mut; 2 – 2 × 3 tables). Separate χ2 analyses were performed to assess the association between the occurrence of neutropenia or of thrombocytopenia and the presence or absence of the mutant gene (2 – 2 × 2 tables). To maintain the overall experiment-wise error rate of α= 0.05, the same degrees of freedom (2) were used to determine the P-value of the χ2 from the 2 collapsed 2 × 2 tables.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Thirty-four dogs were included in this study: 26 ABCB1 wt/wt dogs, 4 ABCB1 wt/mut dogs, and 4 ABCB1 mut/mut dogs (Table 2). The initial χ2 analysis for both neutropenia and thrombocytopenia yielded an association between genotype and neutropenia (P= .0005) as well as genotype and thrombocytopenia (P= .0001). To determine whether or not this association was linked to any dysfunction of P-glycoprotein (ie, presence of the ABCB1-1Δ mutation), dogs of genotype ABCB1 wt/mut and ABCB1 mut/mut were combined into a single group. The 34 dogs in the study thus consisted of 8 dogs in group 1 and 26 in group 2. Group 1 included 7 Collies and 1 Australian Shepherd. Three Collies and 1 Australian Shepherd were ABCB1 wt/mut and 4 Collies were ABCB1 mut/mut. Group 2 dogs (ABCB1 wt/wt) consisted of 5 Border Collies, 5 Shetland Sheepdogs, 3 Australian Shepherds, 1 German Shepherd dog, 1 Collie, 1 Australian Cattle dog, 1 Corgi, and 9 nonherding breed dogs. All dogs received vincristine at a dosage of 0.5–0.7 mg/M2. Four dogs, all ABCB1 wt/wt, received l-asparaginase concurrently with vincristine and did not experience neutropenia or thrombocytopenia. These included a Labrador Retriever, Australian Cattle Dog, Shetland Sheepdog, and Australian Shepherd.

Table 2.   ABCB1 genotype, herding breed status, vincristine dosage range, and hematologic toxicity grade for canine lymphoma patients included in this study.
ABCB1 GenotypeNumber of Dogs and BreedVincristine Dose Range (mg/M2)Neutropenia GradeThrombocytopenia Grade
0123401234
  • a

    All Collies.

  • b

    b 3 Collies, 1 Australian Shepherd.

Mutant/Mutant4 herding breed dogsa0.5–0.722211
Mutant/WT4 herding breed dogsb0.5–0.722112
WT/WT17 herding breed and 9 nonherding breed dogs0.5–0.723111251

For the 8 dogs in group 1, 6/8 (75%) experienced neutropenia and 5/8 (62.5%) experienced thrombocytopenia (Table 2). Four of eight (50%) experienced both neutropenia and thrombocytopenia as a result of vincristine treatment. For the 26 dogs in group 2, 3/26 (11.5%) experienced neutropenia, and 1/26 (4%) experienced thrombocytopenia (this dog also was neutropenic). Interestingly, all three of these dogs were Border Collies.

Dogs with the ABCB1-1Δ mutation (group 1) were significantly more likely to experience neutropenia (P= .00178) and thrombocytopenia (P= .0001) after treatment with vincristine than were ABCB1 wt/wt dogs (group 2). There was no statistical difference between ABCB1 mut/wt dogs and ABCB1 mut/mut dogs in terms of likelihood of developing neutropenia (P= .1025) or thrombocytopenia (.4652) after treatment with vincristine; however, there was very limited statistical power (1 degree of freedom).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study shows the impact of ABCB1-1Δ polymorphism on vincristine-induced hematologic toxicity. The incidence of vincristine-induced hematologic toxicity of any type in a recent study of canine lymphoma was 15.6%,4 which is similar to the results for group 2 dogs (ABCB1 wt/wt) in this study (11.5%). The previous study did not assess ABCB1 genotype, but the authors did not report any Collies, Australian Shepherds, Border Collies, or Shetland Sheepdogs in the study, so it is unlikely that a substantial percentage of dogs in that study consisted of those with the ABCB1-1Δ mutation. Therefore, the fact that 75% of dogs that were either heterozygous or homozygous for the ABCB1-1Δ mutation experienced neutropenia and 62.5% experienced thrombocytopenia is of concern. It is likely that the hematologic toxicity in these patients is a result of vincristine-induced myelosuppression because the nadir of vincristine-induced neutropenia and thrombocytopenia is within the 5–15-day study period.1,3 Additionally, none of the dogs had neutropenia or thrombocytopenia before vincristine treatment.

In addition to myelotoxicity, vincristine can cause gastrointestinal toxicity and neurological toxicity, which often is manifested as a neuropathy. Although gastrointestinal toxicity data were not recorded for all dogs in this study, the medical records of 3 dogs in group 1 and 1 dog in group 2 indicated severe gastrointestinal toxicity (eg, anorexia, vomiting, and diarrhea) associated with vincristine administration. One of the group 1 dogs that experienced gastrointestinal toxicity also developed peripheral neuropathy, which was attributed to vincristine toxicity. Available medical records for 1 dog in group 2 also indicated severe peripheral neuropathy, which was attributed to vincristine (this dog did not have signs of hematologic or gastrointestinal toxicity). Whether or not gastrointestinal or neurological toxicity is more likely to occur in dogs with the ABCB1-1Δ mutation was not determined in this study.

Increased susceptibility to vincristine-induced toxicity in dogs with the ABCB1-1Δ mutation is likely because of impaired excretion of vincristine. The ABCB1 gene codes for P-glycoprotein, a drug transport pump for which vincristine is a substrate. P-glycoprotein is expressed on biliary canalicular cells and renal tubular cells where it is known to excrete substrates into the bile and renal tubular fluid (urine), respectively. Dysfunctional P-glycoprotein, as occurs in dogs with the ABCB1-1Δ mutation, would impair vincristine clearance from the body, resulting in prolonged exposure to vincristine. In a study of rodents, pharmacologic inhibition of P-glycoprotein resulted in a 6-fold reduction in biliary excretion of vincristine.8 Whether or not these rats would have experienced hematologic toxicity is not known because they were euthanized 6 hours after vincristine administration. Three dogs in group 1 received cyclophosphamide, which generally is considered to be more myelosuppressive than vincristine, yet did not experience hematologic toxicity. Cyclophosphamide is not a substrate for P-glycoprotein, and therefore its elimination from the body would not be affected by the ABCB1-1Δ mutation. Two dogs in group 1 also received doxorubicin (a P-glycoprotein substrate) at some point during their treatment. Doxorubicin clearance is also dependent on P-glycoprotein-mediated excretion into bile and urine.13 As would be expected in patients with dysfunctional P-glycoprotein, doxorubicin caused myelosuppression and gastrointestinal toxicity in both of these dogs.

Three dogs in group 2 (ABCB1 wt/wt) developed hematologic toxicity, with 2 dogs developing only neutropenia and 1 dog developing both neutropenia and thrombocytopenia. The dog that experienced both neutropenia and thrombocytopenia also developed sterile hemorrhagic cystitis, myelosuppression, and gastrointestinal toxicity after cyclophosphamide treatment, suggesting generalized impairment of hepatic and renal clearance mechanisms. A single polymorphism most likely cannot predict vincristine toxicity in every patient. Thus, it was not surprising that a few ABCB1 wt/wt patients developed vincristine toxicity. What was surprising, however, was the fact that each of the ABCB1 wt/wt dogs that developed hematologic toxicity was a Border Collie. The authors are not aware of any reports of a breed predilection for vincristine-associated hematologic toxicity in Border Collies. Although this observation is interesting and suggests a breed-related drug sensitivity, these findings must be interpreted cautiously. As stated previously, initial accessions included all dog breeds to ensure an adequate population of ABCB1 wild type dogs, but because the ABCB1-1Δ mutation has been identified only in herding breed dogs, later accessions were limited to herding breeds only. This obviously biased the study population. Additional studies investigating a possible breed predilection for vincristine-induced hematologic toxicity in Border Collies are warranted.

A number of other anticancer drugs, in addition to vincristine, are also substrates for P-glycoprotein. Examples include vinblastine, doxorubicin, taxanes such as paclitaxel, and epipodophyllotoxins such as etoposide.14,15 Whether or not dogs with the ABCB1-1Δ mutation have increased susceptibility to toxic effects of these drugs is not known, but seems likely.

We speculated that even dogs with the ABCB1 wt/mut genotype would experience increased susceptibility to vincristine-induced hematologic toxicity. This speculation was based primarily on a recent case report describing an ABCB1 wt/mut dog that was highly sensitive to vincristine and doxorubicin hematologic and gastrointestinal toxicity (both at normal and decreased dosages), but was able to tolerate a standard dosage of cyclophosphamide.10 The results of the current study appear to corroborate the findings in the case report. One would assume, based on these results, that dogs with the ABCB1 wt/mut genotype have deficient P-glycoprotein function with respect to biliary and renal excretion of vincristine. Whether or not ABCB1 wt/mut and ABCB1 mut/mut dogs would have the same dose-toxicity profile for vincristine is not known. Evaluation of greater numbers of ABCB1 mut/wt and ABCB1 wt/wt patients may have yielded a statistically significant difference between groups.

In conclusion, this study supports the clinical utility of pharmacogenetics in dogs with cancer. Seven of 8 (87.5%) dogs that harbored the ABCB1-1Δ mutant allele (either homozygous or heterozygous) experienced vincristine-induced hematologic toxicity in comparison to only 11% of normal (ie, ABCB1 wt/wt) dogs. Identifying patients that harbor the ABCB1-1Δ mutation before treatment may be used to adjust vincristine dosage, avoiding hematologic toxicity and enhancing efficacy by preventing treatment delays. In contrast, using a lower dosage of vincristine in all herding breed dogs, regardless of genotype, would result in subtherapeutic dosing in a substantial number of patients. Subtherapeutic dosing of vincristine could result in lower remission rates and decreased remission duration. Breeds that have been documented to harbor the ABCB1-1Δ mutation include several herding breeds and 2 sight hound breeds (Table 3). Additional studies are required to determine appropriate dosages of vincristine for patients with the ABCB1-1Δ mutation.

Table 3.   Dog breeds and allelic frequency of the ABCB1-1Δ mutation.16,17
BreedAllele, % ABCB1-1Δ
Australian Shepherd16.6
Australian Shepherd, Miniature25.9
Border Collie0.6
Collie54.6
English Shepherd Dog7.1
German Shepherd Dog6.0
Herding Breed Cross6.0
Long-haired Whippet41.6
McNab17.1
Old English Sheepdog6.3
Shetland Sheepdog8.4
Silken Windhound17.9
Swiss Shepherd Dog13.0

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was funded by proceeds from the Veterinary Clinical Pharmacology Laboratory, College of Veterinary Medicine, Washington State University.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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    Mechetner E, Kyshtoobayeva A, Zonis S, et al. Levels of multidrug resistance (MDR1) P-glycoprotein expression by human breast cancer correlate with in vitro resistance to taxol and doxorubicin. Clin Cancer Res 1998;4:389398.
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    Silverman JA. Multidrug-resistance transporters. Pharm Biotechnol 1999;12:353386.
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    Geyer J, Doring B, Godoy JR, et al. Frequency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany. J Vet Pharmacol Ther 2005;28:545551.
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    Neff MW, Robertson KR, Wong AK, et al. Breed distribution and history of canine mdr1-1Delta, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage. Proc Natl Acad Sci USA 2004;101:1172511730.