* Objective.-To evaluate the occurrence of megaloblastic anemia induced by the infusion of therapeutic or prophylactic methotrexate in patients with acute leukemia.
Design.-Data on 3 patients with acute leukemia receiving intrathecal methotrexate were prospectively analyzed.
Setting.-Large tertiary-care center.
Results.-All 3 patients with acute leukemia developed megaloblastic anemia confirmed by examination of the bone marrow aspirate and biopsy. Two of the 3 patients had low folic acid levels, while all patients had normal serum B12 levels. All patients responded favorably to a therapeutic trial of folic acid. The median time for recovery of the hematologic parameters in these patients was 7 days.
Conclusions.-Intrathecally administered methotrexate may result in megaloblastic changes in the bone marrow of leukemic patients. The morphologic clues suggestive of folate deficiency in patients with acute leukemia may be masked by coexisting factors, such as the effects of cytotoxic treatment, prior transfusions, or persistent changes from the leukemic clone itself. Caution should be exercised to avoid attributing these changes to the neoplastic process, since the prognosis and treatment for the conditions involved are totally different. Repeat examination of the bone marrow, obtaining folic acid and vitamin B12 levels, and a therapeutic trial of folic acid may help identify and reverse these changes.
(Arch Pathol Lab Med. 1999;123:774-777)
By binding critical enzymes, folate acts as a cofactor and functions to receive a single-carbon fragment from selected enzymatic reactions and transfer this carbon unit to other molecules important for de novo synthesis of purines and pyrimidines, precursors of DNA.1 To form an active compound, folate must be reduced to tetrahydrofolate (FH^sub 4^). The enzyme dihydrofolate reductase, a critical enzyme in intracellular methylation reactions, catalyzes the reduction of folic acid to dihydrofolate and FH^sub 4^.1,2
Methotrexate (MTX), an inhibitor of dihydrofolate reductase, is often used in the treatment of hematologic malignancies, as well as for prophylaxis and treatment of the central nervous system in patients with acute leukemia or lymphoma. When used intravenously or orally, MTX can cause depletion of intracellular tetrahydrofolate, leading to megaloblastic anemia.
In MTX-treated cells, inhibition of dihydrofolate reductase results in the entrapment of folate as inactive dihydrofolate with subsequent suppression of thymidylate synthesis, leading to a reduction in the rate of DNA synthesis.3,4 The slowing of DNA synthesis in combination with normal cytoplasmic RNA synthesis leads, over several cell divisions, to asynchrony between the rates of cytoplasmic and nuclear maturation. This unbalanced growth affects all proliferating cells, but is most easily observed in bone marrow cells, which morphologically appear to be megaloblastic.
REPORT OF CASES
The following is a critical description of the courses of 3 patients with acute leukemia and megaloblastic anemia. The Table provides a detailed laboratory analysis of each case before and after folic acid supplementation and lists each patient's MTX, folic acid, and vitamin B12 level at the time megaloblastic anemia was diagnosed.
A 64-year-old woman with acute myelomonocytic leukemia (French-American-British grade M4), presented 2 months after the end of her consolidation treatment with headache, confusion, and increasing fatigue. Cerebrospinal fluid (CSF) cytology revealed numerous blasts consistent with leukemic involvement. Bone marrow aspirate and biopsy revealed no evidence of leukemia. Methotrexate at a dose of 15 mg was administered intrathecally via lumbar puncture and then through ventricular reservoir for 2 additional doses. Repeat CSF analysis showed normal protein and glucose levels and no blasts. Two weeks later, the patient presented with increasing fatigue and mild chest discomfort. Laboratory analysis at the time demonstrated an absolute neutrophil count of 310/(mu)L; platelets, 14 x 10^sup 9^/L; hemoglobin, 88 g/L; and mean cell volume, 95.5 fL. Review of the blood film revealed nuclear hypersegmentation of neutrophils, as depicted in Figure 1. Repeat examination of the bone marrow aspirate and biopsy showed megaloblastic erythroid hyperplasia and large metamyelocytes (Figure 2). The patient's lactate dehydrogenase (LDH) level was 296 U/L (normal, 122-240 U/L). After obtaining folic acid and vitamin B12 levels, the patient was given a therapeutic trial of 5 mg folic acid daily; white blood cell and platelet counts recovered on day 7, and LDH levels normalized by day 21.
A 20-year-old woman with acute lymphoblastic leukemia underwent induction treatment with L-asparaginase, doxorubicin, vincristine, prednisone, cytosine-arabinoside, and cyclophosphamide. Bone marrow aspirate and biopsy following treatment showed no evidence of leukemia. Two weeks later, consolidation treatment with vincristine and doxorubicin was started, and prophylactic intrathecal MTX was administered at a dose of 12 mg via lumbar puncture twice weekly. Repeat complete blood counts following the third dose of MTX showed an absolute neutrophil count of 1210/(mu)L; platelets, 19 x 10^sup 9^/L; hemoglobin, 74 g/L; and mean cell volume, 112 fL. Few ovalocytes and anisopoikilocytosis were present in the peripheral blood smear. This degree of pancytopenia was disproportionate to what was expected from this particular chemotherapy regimen. Therefore, we examined the bone marrow aspirate and biopsy, which revealed megaloblastic erythroid hyperplasia and giant metamyelocytes. The patient's LDH level at this presentation was 310 U/L. After obtaining folic acid and vitamin B12 levels, a therapeutic trial of 2-mg daily doses of folic acid was started. No complete blood count was available on this patient on day 7; however, repeat laboratory analysis showed improvement in both the hematologic parameters and LDH by day 14 of folic acid replacement.
The patient, a 29-year-old man with acute lymphoblastic leukemia in remission, verified by bone marrow examination, was started on prophylactic administration of MTX at a dose of 12 mg through ventricular reservoir. Following the third dose, the patient presented with increasing fatigue and shortness of breath. Laboratory findings at the time of presentation showed the patient's absolute neutrophil count to be 940/(mu)L; platelets, 22 x 10^sup 9^/ L; hemoglobin, 79 g/L; and mean cell volume, 103 fL. Mild anisocytosis, poikilocytosis, and few tear drops were evident in the blood film. Examination of the bone marrow aspirate and biopsy showed megaloblastic erythroid hyperplasia. The patient's LDH level was 276 U/L. After obtaining folic acid and B12 levels, a daily replacement with 2 mg folic acid was administered, resulting in improvement of the blood counts by day 7 and normalization of LDH by day 14.
MATERIALS AND METHODS
Folate and Vitamin B12
Folate and vitamin B12 were measured on a Technicon Immuno 1 (Technicon, Tarrytown, NY) system using competitive magnetic separation immunoassays. Folate and vitamin B12 were first released from serum binders with a pretreatment. The released vitamins were reacted with a binding protein reagent (folate binding protein and intrinsic factor for folate and vitamin B12, respectively) and incubated at 37 deg C. Folate or B12 conjugated to an alkaline phosphatase enzyme was then added to compete with the sample for the binding protein. A monoclonal immunomagnetic particle was incubated with the reagents, washed, and paranitrophenyl phosphate substrate was added. Absorbance at 405 nm due to the formation of paranitrophenoxide is inversely proportional to the amount of vitamin in the sample. The folate assay is linear from 0.7 to 34 nmol/L (0.3-15 ng/mL), with a normal range of 5.9 to 39.2 nmol/L (2.6-17.3 ng/mL) for serum folate and 362.6 to 1602.1 nmol/L (160-707 ng/mL) for red blood cell folate; the vitamin B12 assay is linear from 19 to 1106 pmol/L (26-1500 pg/mL), with a normal range of 182 to 672 pmol/L (247-911 pg/mL). These tests were performed after examining the bone marrow aspirate and biopsy, and before replacement with folic acid.
Methotrexate was measured on a TDX analyzer (Abbott Laboratories USA, Abbott Park, Ill) using a fluorescence polarization immunoassay. This technique adds a pretreatment reagent to free any bound analyte from serum proteins. A specific monoclonal antibody is added together with a tracer-labeled MTX that competes for the antibody with the MTX in the sample. The intensity of the fluorescent signal varies inversely with the amount of MTX in the sample. The assay range is 0 to 1000 (mu)mol/L with a lower limit of detection of 0.05 (mu)mol/L. Methotrexate concentrations were measured 48 to 72 hours after treatment.
Bone Marrow Preparation
Bone marrow biopsies were fixed for 2 hours in 10% buffered formalin, decalcified for 30 minutes in Baxter Decal, and processed routinely for surgical pathology. Biopsies were evaluated for cellularity; cellular composition, including the presence or absence of clusters of leukemic blasts; and the overall adequacy of megakaryocytes. Aspirate particle smears and biopsy touch preparations were stained with Wright-Giemsa stain and were examined to determine the myeloid-erythroid ratio, evaluate trilineage hematopoiesis, and evaluate the presence or absence of leukemic blasts. Storage iron was evaluated with Prussian blue stain. Special stains for myeloperoxidase, chloroacetate esterase, and (alpha)-naphthyl butyrate esterase and immunophenotyping by flow cytometry were performed as needed to determine leukemic lineage.
Follow-up complete blood count and LDH values were performed on a weekly basis after the administration of folic acid supplements until normalization of these parameters and then as dictated by each patient's treatment plan. Laboratory data at 1 week posttreatment were not available for patient 2, owing to noncompliance with our instructions.
Intrathecally administered MTX resulted in megaloblastic anemia in 3 patients with acute leukemia. None of these patients had morphologic evidence of relapsed leukemia in their bone marrow biopsies at the time of assessment.
Serum folate levels were below normal in patients 2 and 3, but were normal in patient 1. Serum B12 levels were normal in all patients. Methotrexate concentrations were below detectable limits in all patients. Replacement with folic acid led to improvement of the hematologic parameters and LDH in all patients. None of the patients had evidence of systemic toxicity or end-organ damage at the time of administration.
Methotrexate is an antineoplastic agent used in the treatment of several malignancies. Through its tight binding of dihydrofolate reductase, MTX inhibits the production of tetrahydrofolates necessary for tumor cell growth. Methotrexate exhibits activity in leukemia and lymphoma, two malignancies with a propensity for sequestration in the central nervous system.
Owing to its low lipid solubility, diffusion of MTX into the CSF in concentrations necessary for tumor cell kill (usually 1 (mu)mol/L) are not achievable at "conventional" doses in the range of 30 to 100 mg/m^sup 2^ (CSF-plasma ratio, 0.03). This obstacle may be overcome by (1) administering the agent in higher doses (eg, >=1 g/m^sup 2^) or (2) direct administration into the CSE The expected corollary is that intrathecally administered MTX can slowly diffuse from the CSF into the systemic circulation, yielding prolonged elevated plasma levels and potential toxicities.5
Elimination from the central nervous system is primarily by bulk resorption of CSF Jacobs and colleagues6 compared the pharmacokinetics of orally, intravenously, and intrathecally administered MTX in 2 patients with acute lymphocytic leukemia. Their results suggest that MTX given intrathecally slowly diffuses into the plasma, leading to concentrations greater than 10^sup -8^ mol/L for twice as long as expected for the same dose given orally or intravenously. A patient with high-grade lymphoma and central nervous system involvement who developed tumor lysis after 1 dose of intrathecal MTX has also been described.7
In this short series, 3 patients received intrathecal MTX and subsequently developed megaloblastic bone marrow changes and peripheral cytopenias that recovered following replacement with folic acid. The course of improvement of both the peripheral counts (average, 7 days) and LDH (average, 14 days) corresponds well with the reversion of the effects of folic acid deficiency following replacement. All the patients had serum MTX levels below the limits of detection, suggesting that there was no laboratory evidence of systemic toxicity. In support of this finding is the absence of clinical symptoms or signs related to MTX toxicity, such as nausea, vomiting, and mucositis.
As was noted, patient 1 had normal folic acid and vitamin B12 values. However, examination of the bone marrow and peripheral blood smear in this patient demonstrated findings consistent with megaloblastic anemia, including the multilobated neutrophils depicted in Figure 1. In addition, the patient had a well-documented response to a therapeutic trial of folic acid.
It is important to realize that none of the patients had renal insufficiency or third space fluid collection, factors that may prolong the exposure to MTX and lead to systemic toxicity. Also, none of the patients at the time of their evaluation was receiving other medications that might have induced folic acid deficiency.
It appears that intrathecally administered MTX may have resulted in megaloblastic bone marrow changes by a diffusion mechanism from the central nervous system to the systemic circulation in amounts not sufficient to cause systemic toxicity. An alternative explanation is that our 3 previously treated patients had borderline low folic acid levels, and that even a small leakage of MTX to the systemic circulation led to megaloblastic anemia.
Also noteworthy were the extreme neutropenia and thrombocytopenia observed in the 3 patients, as well as the lack of the typical hypercellular marrow characteristic of nutritional megaloblastic anemia.8 These findings may be explained by prior exposure to systemic chemotherapy and the persistence of a subclinical leukemic clone.
It is crucial not to assume automatically that pancytopenia in the peripheral blood or megaloblastic erythropoiesis in the bone marrow of patients receiving intrathecal MTX is due to relapsed leukemia, since these findings may also be due to MTX administration and may respond favorably to folic acid therapy.
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2. Steinberg SE, Fonda S, Campbell CL, Hillman RS. Cellular abnormalities of folate deficiency. Br J Haematol. 1983;54:605-612.
3. Jolivet J, Chabner BA. Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells: selective retention and less dissociable bindings of 4-N-10-CH^sub 3^-pterolglutamate^sub 4^ and 4-NH^sub 2^-10-CH^sub 3^--pterolglutamate, to dihydrofolate reductase. J Clin Invest.1983;72:773-778.
4. Bleyer WA. The clinical pharmacology of methotrexate: new applications of an old drug. Cancer. 1978;41:36-51.
5. Bleyer WA, Nelson A, Kamen BK. Accumulation of methotrexate in systemic tissues after intrathecal administration. J Pediatr Hematol Oncol. 1997;19:530532.
6. Jacobs SA, Bleyer WA, Chabner BA, Johns DG. Altered plasma pharmacokinetics of methotrexate administered intrathecally. Lancet. 1975;1:455-456.
7. Simmons ED, Somberg KA. Acute tumor lysis syndrome after intrathecal methotrexate administration. Cancer.1991;57:2062-2065.
8. Easton DJ. Severe thrombocytopenia associated with acute folic acid deficiency and severe hemorrhage in two patients. Can Med Assoc J 1984;130:418420.
Accepted for publication March 1, 1999.
From the Department of Medicine, Division of Hematology/Oncology (Dr Sallah), Department of Pathology and Laboratory Medicine (Dr Hanrahan), and Leo Jenkins Cancer Center (Dr Phillips), East Carolina University, School of Medicine, Greenville, NC. Dr Sallah is now with the Department of Medicine, Division of Hematology and Oncology, University of Tennessee, Memphis.
Reprints not available from the author.
Copyright College of American Pathologists Sep 1999
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