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Difficulty measuring methotrexate in a patient with high-dose methotrexate-induced nephrotoxicity.

CASE

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 [micro]mol/L at 24 h after infusion, 450 [micro]mol/L at 48 h, and 350 [micro]mol/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 MTXbelowthe toxic concentration, however, and the decision was made to give the patient glucarpidase [carboxypeptidase [G.sub.2] ([CPDG.sub.2]); 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 [CPDG.sub.2] administration and diluted with 9 volumes of diluent (10-fold dilution) was 9.6 [micro]mol/L, whereas the measured concentration was 50 [micro]mol/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 [CPDG.sub.2] 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 [micro]mol/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 [micro]mol/L.

DISCUSSION

HDMTX, defined as MTX doses [greater than or equal to] 1000 mg/[m.sup.2] 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 [micro]mol/L at 24 h, >1 [micro]mol/L at 48 h, or >0.1 [micro]mol/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.

[CPDG.sub.2] is a bacterial enzyme available in a recombinant form cloned from Pseudomonas strain RS-16. [CPDG.sub.2] rapidly hydrolyzes the C-terminal glutamate from MTX to form the inactive compounds 2,4diamino-[N.sup.10]-methylpteroic acid (DAMPA) and glutamate, thus providing a rapid route of elimination. [CPDG.sub.2] 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. [CPDG.sub.2] administration is recommended for patients whose plasma MTX concentration is >10 [micro]mol/L by 42-48 h after beginning MTX infusion (2). The patient in this case clearly met the criteria for receiving [CPDG.sub.2].

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 [CPDG.sub.2] 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 [CPDG.sub.2] 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 [CPDG.sub.2] 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 [CPDG.sub.2]. To confirm the DAMPA interference and to have the ability to accurately measure MTX concentrations after [CPDG.sub.2] 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 [CPDG.sub.2] dose was effective in reducing the plasma MTX concentration by 99% (from 230 [micro]mol/L to 2.2 [micro]mol/L). After DAMPA clearance from the patient's circulation by 7 days after [CPDG.sub.2] administration, MTX concentrations measured by FPIA were comparable to those obtained by LC-MS/MS (Fig. 1).

[FIGURE 1 OMITTED]

QUESTIONS TO CONSIDER

1. What is the incidence of MTX-induced nephrotoxicity, and how is it treated?

2. What is the mechanism of [CPDG.sub.2] action, and what is its clinical utility?

3. How is MTX measured, and what is the source of the discrepancy in the patient's MTX measurements?

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.

* [CPDG.sub.2] 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 [CPDG.sub.2] and the inability of immunoassays to accurately measure MTX in [CPDG.sub.2]-treated patients.

* It is critical that the clinical team notify the laboratory when [CPDG.sub.2] is administered to avoid the reporting of falsely increased MTX concentrations that would indicate failure of [CPDG.sub.2] therapy.

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.

References

(1.) Erttmann R, Bielack S, Landbeck G. Kinetics of 7-hydroxy-methotrexate after high-dose methotrexate therapy. Cancer Chemother Pharmacol 1985; 15:101-4.

(2.) Widemann BC, Adamson PC. Understanding and managing methotrexate nephrotoxicity. Oncologist 2006;11:694-703.

(3.) Widemann BC, Balis FM, Kempf-Bielack B, Bielack S, Pratt CB, Ferrari S, et al. High-dose methotrexate-induced nephrotoxicity in patients with osteosarcoma. Cancer 2004;100:2222-32.

(4.) Kuo CY, Wu HL, Kou HS, Chiou SS, Wu DC, Wu SM. Simultaneous determination of methotrexate and its eight metabolites in human whole blood by capillary zone electrophoresis. J Chromatogr A 2003;1014:93-101.

(5.) Widemann BC, Balis FM, Adamson PC. Dihydrofolate reductase enzyme inhibition assay for plasma methotrexate determination using a 96-well microplate reader. Clin Chem 1999;45:223-8.

(6.) Donehower RC, Hande KR, Drake JC, Chabner BA. Presence of 2,4-diamino-[N.sup.10]-methylpteroic acid after high-dose methotrexate. Clin Pharmacol Ther 1979;26:63-72.

(7.) Albertioni F, Rask C, Eksborg S, Poulsen JH, Pettersson B, Beck O, et al. Evaluation of clinical assays for measuring high-dose methotrexate in plasma. Clin Chem 1996;42:39-44.

(8.) Pesce MA, Bodourian SH. Evaluation of a fluorescence polarization immunoassay procedure for quantitation of methotrexate. Ther Drug Monit 1986;8:115-21.

(9.) Kumar VS, Law T, Kellogg M. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for monitoring methotrexate in the setting of carboxypeptidase-G2 therapy. In: Garg U, Hammett-Stabler CA, eds. Clinical applications of mass spectrometry: methods and protocols. New York: Springer; 2009. p 359-63. Methods in molecular biology, vol 603.

Commentary

Elizabeth Fox * and Frank M. Balis

High-dose methotrexate-induced nephrotoxicity is a medical emergency. Renal methotrexate excretion, which typically accounts for 90% of the drug's elimination, is delayed, resulting in prolonged exposure to high methotrexate concentrations. The duration of exposure is the primary determinant of the drug's toxic effects, and early recognition and prompt efforts to lower methotrexate concentrations are critical to preventing severe systemic toxicity. High-dose methotrexate-induced renal dysfunction is heralded by an increasing serum creatinine concentration during or shortly after the methotrexate infusion. Urine output is usually maintained despite a rapid decline in glomerular filtration. Daily monitoring of serum creatinine and methotrexate concentrations is essential to early detection of this complication.

Leucovorin provides a source of the tetrahydrofolates that are depleted by methotrexate's inhibition of dihydrofolate reductase, but methotrexate competes with leucovorin for cell uptake. Therefore, leucovorin rescue is less effective at methotrexate concentrations that exceed 10 [mu]mol/L for 48 h. The leucovorin dose must be increased in proportion to the serum methotrexate concentration when methotrexate clearance is delayed (e.g., 1000 mg/[m.sup.2] every 6 h for a methotrexate concentration [greater than or equal to] 10 [mu]mol/L at 48 h). High leucovorin doses (250 mg/[m.sup.2] every 6 h) should also be continued for 48 h after glucarpidase administration because the enzyme hydrolyzes leucovorin and its active circulating metabolite, 5-methyltetrahydrofolate, to inactive forms.

Glucarpidase rapidly and efficiently lowers the serum methotrexate concentration by providing an alternative route of elimination and, when administered as soon as possible after the recognition of nephrotoxicity, can effectively prevent methotrexate toxicity. Patients who receive inadequate leucovorin rescue or receive glucarpidase >96 h after the start of the methotrexate infusion are at greater risk for developing life-threatening methotrexate toxicity (1).

As illustrated by the case study, commercial methotrexate assays will underestimate the impact of glucarpidase on serum methotrexate concentrations because of the interference by the inactive byproduct, DAMPA. DAMPA is subsequently metabolized by hydroxylation and glucuronide conjugation and is cleared more rapidly than residual methotrexate.

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.

Reference

(1.) Widemann BC, Balis FM, Kim A, Boron M, Jayaprakash N, Shalabi A, et al. Glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction: clinical and pharmacologic factors affecting outcome. J Clin Oncol 2010;28:3979-86.

Elizabeth Fox * and Frank M. Balis

Division of Oncology and Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, PA.

* Address correspondence to this author at: Division of Oncology and Center for Childhood Cancer Research, CTRB-4016, The Children's Hospital of Philadelphia, 3501 Civic Center Blvd., Philadelphia, PA 19104. Fax 267425-01 13; e-mail foxe@email.chop.edu.

Received September 17, 2010; accepted September 20, 2010.

DOI: 10.1373/clinchem.2010.153601

Commentary

Michael C. Milone *

Glucarpidase (Voraxaze[R]), currently an investigational agent available under an open-label treatment protocol, offers great potential for the treatment of patients with or at high risk for toxicity after high-dose methotrexate (MTX) chemotherapy. The case study reported by Al-Turkmani et al. illustrates the challenges to the laboratory monitoring of MTX, especially in the glucarpidase-treated patient. Metabolite interference has long been recognized in MTX immunoassays. Although a relatively small positive bias is due to interference by 7-hydroxymethotrexate, the predominant metabolite of MTX, this case demonstrates the appreciable and unpredictable nature of metabolite interference. The high positive bias reported for this case caused by the typically minor MTX metabolite 2,4-diamino-N10methylpteroic acid (DAMPA) rendered monitoring of MTX useless for the management of the patient. Even with the astute recognition of an analytical interference by the authors' laboratory, the patient was still subject to an additional glucarpidase dose that might have been unnecessary. Failure to recognize the interference could have lead to even further, potentially harmful interventions. I doubt other laboratories would have fared better in detecting this interference. The availability of a more specific liquid chromatography method for MTX is unlikely in most clinical laboratories. Proficiency-testing data from the College of American Pathologists for 2010 reveal that 92% of laboratories perform the fluorescence polarization immunoassay used in this case, and all of the 407 participating laboratories currently use an immunoassay platform for MTX. As Al-Turkmani et al. state, it is imperative that laboratory directors communicate with their oncology colleagues and educate them about the limitations of MTX assays, especially for the glucarpidase-treated patient. The limitations of MTX concentration monitoring should also be considered for inclusion in the package insert for glucarpidase to help disseminate this important information.

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.

Toxicology/TDM Laboratory, Hospital of the University of Pennsylvania, Philadelphia, PA.

Michael C. Milone *

* Address correspondence to the author at: Hospital of the University of Pennsylvania, Founders 7.103, 3400 Spruce St., Philadelphia, PA 19104. Fax 215662-7529; e-mail milone@mail.med.upenn.edu.

Received September 13, 2010; accepted September 20, 2010.

DOI: 10.1373/clinchem.2010.153619

M. Rabie Al-Turkmani, [1] Terence Law, [1] Anupama Narla, [2] and Mark D. Kellogg [1] *

[1] Department of Laboratory Medicine, Children's Hospital Boston, Boston, MA;

[2] Dana Farber Cancer Institute, Children's Hospital Boston, and Harvard Medical School, Boston, MA.

[3] Nonstandard abbreviations: HDMTX, high-dose methotrexate; MTX, methotrexate; [CPDG.sub.2], carboxypeptidase G2; DAMPA, 2,4-diamino-[N.sup.10]-methylpteroic acid; FPIA, fluorescence polarization immunoassay; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

* Address correspondence to this author at: Department of Laboratory Medicine, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115. Fax 617-730-0383; e-mail mark.kellogg@childrens.harvard.edu.

Received February 4, 2010; accepted June 15, 2010.

Previously published online at DOI: 10.1373/clinchem.2010.144824
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Title Annotation:Clinical Case Study
Author:Turkmani, M. Rabie Al-; Law, Terence; Narla, Anupama; Kellogg, Mark D.
Publication:Clinical Chemistry
Date:Dec 1, 2010
Words:3156
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