An 18-year-old male presented with pain and swelling in his left leg that he noted after playing football. An x-ray of the affected leg showed a destructive lesion that prompted a concern for malignancy. Subsequent tests, including magnetic resonance imaging, a bone scan, and a needle biopsy of the lesion, confirmed nonmetastatic osteosarcoma in the left proximal tibia. The patient was started on a standard regimen of chemotherapy. He received 4 cycles of high-dose methotrexate (HDMTX)3 with leucovorin rescue and 2 cycles of cisplatin and doxorubicin, which he tolerated well. Each HDMTX course involved the intravenous administration of 20 g methotrexate (MTX) over 4 h. The patient experienced delayed MTX clearance after the first cycle but showed typical clearance after the subsequent 3 cycles. He then underwent a planned radical resection of the tumor with allograft placement. After the patient recovered from surgery, chemotherapy resumed, and the patient received 2 additional cycles of cisplatin and doxorubicin and 1 additional cycle of HDMTX. His treatment was interrupted when he had to undergo surgery for a wound infection in the affected leg. After recovery from the second surgery, the patient received a sixth HDMTX cycle. After this cycle, the patient developed acute nephrotoxicity, which was manifested by marked renal dysfunction and delayed MTX clearance. His plasma creatinine concentration increased from 0.8 mg/dL (8 mg/L) at the start of the cycle to 6.8 mg/dL (68 mg/L) after he received HDMTX. Plasma MTX concentrations were 1700 μmol/L at 24 h after infusion, 450 μmol/L at 48 h, and 350 μmol/L at 72 h. The patient was treated with aggressive hydration, diuresis, and 1500 mg leucovorin intravenously every 6 h for several days. These measures did not reduce MTX below the toxic concentration, however, and the decision was made to give the patient glucarpidase [carboxypeptidase G2 (CPDG2); BTG International] 4 days after he received the HDMTX infusion.
The laboratory experienced difficulty in reporting subsequent plasma MTX concentrations because of discrepancies in the Abbott TDx immunoassays of the MTX concentrations in serially diluted samples. For example, the plasma MTX concentration for a sample obtained after CPDG2 administration and diluted with 9 volumes of diluent (10-fold dilution) was 9.6 μmol/L, whereas the measured concentration was 50 μmol/L for the same sample diluted with 99 volumes of diluent (100-fold dilution).
Because MTX could not be measured accurately and because of concern for ongoing MTX toxicity, a second CPDG2 dose was administered to the patient 2 days after the first. Five days later, the discrepancy in MTX measurements disappeared, and the laboratory was able to report subsequent plasma MTX concentrations, which were <4.5 μmol/L. Because MTX and creatinine concentrations were decreasing steadily, the decision was made to complete intravenous hydration at 170 mL/h and leucovorin rescue with 250 mg administered intravenously every 6 h at home until the MTX concentration was <0.1 μmol/L.
QUESTIONS TO CONSIDER
What is the incidence of MTX-induced nephrotoxicity, and how is it treated?
What is the mechanism of CPDG2 action, and what is its clinical utility?
How is MTX measured, and what is the source of the discrepancy in the patient's MTX measurements?
HDMTX, defined as MTX doses ≥1000 mg/m2 administered by prolonged intravenous infusion followed by leucovorin rescue, has been widely used in the treatment of malignancies such as osteosarcoma, acute lymphoblastic leukemia, and lymphoma. MTX is metabolized primarily to 7-hydroxymethotrexate, the plasma concentrations of which exceed those of the parent compound shortly after HDMTX infusion (1). MTX-induced acute nephrotoxicity is thought to be due to the precipitation of MTX or its insoluble metabolites in the renal tubules or to a direct toxic effect of MTX on the tubules (2). Renal dysfunction causes delayed MTX elimination and unsuccessful rescue by leucovorin. Nephrotoxicity has been reported in clinical trials in approximately 1.8% of patients with osteosarcoma treated with HDMTX. The mortality rate is 4.4% among these patients (3). MTX concentrations >10 μmol/L at 24 h, >1 μmol/L at 48 h, or >0.1 μmol/L at 72 h after infusion are associated with a high risk for nephrotoxicity (2).
HDMTX-induced nephrotoxicity is conventionally managed through hydration and alkalinization of the urine to enhance the solubility and urinary excretion of MTX. Other measures include monitoring of blood creatinine and MTX concentrations, as well as pharmacokinetically guided leucovorin rescue to restore intracellular folate concentrations. Dialysis has also been used for MTX removal.
CPDG2 is a bacterial enzyme available in a recombinant form cloned from Pseudomonas strain RS-16. CPDG2 rapidly hydrolyzes the C-terminal glutamate from MTX to form the inactive compounds 2,4-diamino-N10-methylpteroic acid (DAMPA) and glutamate, thus providing a rapid route of elimination. CPDG2 administration in combination with thymidine and leucovorin is highly effective, decreasing plasma MTX concentration by 95%–99% within 15 min in patients with HDMTX-induced nephrotoxicity (4, 5).
In a patient with an increased plasma MTX concentration and poor renal function, it is crucial to use a rapid means of eliminating MTX from the circulation to prevent further renal damage and to avoid other toxicities, such as myelosuppression and mucositis, that are associated with increased MTX concentrations. CPDG2 administration is recommended for patients whose plasma MTX concentration is >10 μmol/L by 42–48 h after beginning MTX infusion (2). The patient in this case clearly met the criteria for receiving CPDG2.
Plasma MTX concentrations are routinely monitored after HDMTX treatment to determine the rate of drug clearance and the leucovorin dose required for rescue. The method most commonly used for routine MTX measurement in clinical laboratories is fluorescence polarization immunoassay (FPIA). In addition to immunoassays, capillary zone electrophoresis and HPLC methods have been described for measuring MTX and its metabolites in biological fluids (4). A dihydrofolate reductase enzyme-inhibition assay that uses a 96-well plate reader to measure the plasma MTX concentration has also been introduced (5).
Because of the limited analytical range of the MTX assay used in our laboratory and the wide range of concentrations observed in our patients, our standard operating procedure involves the serial dilution (10-fold, 100-fold, 500-fold) of samples with saline. The discrepancy in this patient's serially diluted MTX measurements was due to differing concentrations of the interfering MTX metabolite DAMPA, which was present at a high concentration after CPDG2 treatment. The package insert for the MTX assay states that when MTX and DAMPA are both present, the interference from DAMPA is less (an approximately 26% false increase) than when only DAMPA is present (up to a 59% false increase). Thus, the results for the 10-fold dilutions in this case back-calculated to lower MTX concentrations than those for the 100-fold dilutions because the DAMPA interference was reduced by the presence of MTX. The amount of MTX present in the 100-fold dilutions was well below the linear interval of the assay and allowed greater interaction between the antibody and DAMPA, thereby increasing the observed MTX concentration. This variable interference further limits the utility of FPIA after CPDG2 administration.
DAMPA is typically a minor metabolite of MTX, and its plasma concentration after HDMTX infusion is usually very low (6). The DAMPA interference of MTX immunoassays is well established, however. The MTX FPIA has shown 82.6% cross-reactivity with DAMPA in the method that uses polyclonal antibodies and 41.1% cross-reactivity in the method that uses monoclonal antibodies (7). DAMPA interference causes marked MTX overestimation and makes immunoassays unreliable for MTX measurement after CPDG2 treatment. Unlike DAMPA, the cross-reactivity of 7-hydroxymethotrexate with the MTX FPIA is only 0.6% (8). In the MTX FPIA, one would expect similar low cross-reactivity with the hydroxylated metabolite of DAMPA and the glucuronide of that molecule; however, the degree of interference may be different in polyclonal antibody–based or other forms of the immunoassay.
After the clinical laboratory staff suspected interference in the MTX assay, they contacted the medical team and learned that the patient had received CPDG2. To confirm the DAMPA interference and to have the ability to accurately measure MTX concentrations after CPDG2 administration, we subsequently developed a liquid chromatography– tandem mass spectrometry (LC-MS/MS) method for MTX measurement (9). Use of this LC-MS/MS method to measure MTX concentrations in samples from the presented patient confirmed the DAMPA interference in the FPIA: Plasma MTX concentrations obtained by LC-MS/MS were markedly lower than those obtained with the TDx analyzer with a 10-fold dilution (Fig. 1). LC-MS/MS measurements of MTX also indicated that the first CPDG2 dose was effective in reducing the plasma MTX concentration by 99% (from 230 μmol/L to 2.2 μmol/L). After DAMPA clearance from the patient's circulation by 7 days after CPDG2 administration, MTX concentrations measured by FPIA were comparable to those obtained by LC-MS/MS (Fig. 1).
POINTS TO REMEMBER
Nephrotoxicity has been reported in approximately 1.8% of patients with osteosarcoma treated with HDMTX in clinical trials, with a mortality rate of 4.4% among these patients.
HDMTX-induced nephrotoxicity is conventionally managed through hydration and alkalinization of the urine to enhance the solubility and urinary excretion of MTX. Dialysis has also been used for MTX removal.
CPDG2 rapidly hydrolyzes the C-terminal glutamate of MTX to form the inactive metabolite DAMPA and glutamate, thus providing a rapid route of elimination.
The method most commonly used for routinely measuring MTX in clinical laboratories is FPIA. The DAMPA metabolite exhibits high cross-reactivity with most MTX immunoassays.
The laboratory should educate physicians about the interference caused by CPDG2 and the inability of immunoassays to accurately measure MTX in CPDG2-treated patients.
It is critical that the clinical team notify the laboratory when CPDG2 is administered to avoid the reporting of falsely increased MTX concentrations that would indicate failure of CPDG2 therapy.
↵3 Nonstandard abbreviations:
- high-dose methotrexate;
- carboxypeptidase G2;
- 2,4-diamino-N10-methylpteroic acid;
- fluorescence polarization immunoassay;
- liquid chromatography–tandem mass spectrometry
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
- Received for publication February 4, 2010.
- Accepted for publication June 15, 2010.
- © 2010 The American Association for Clinical Chemistry