Methotrexate-induced pancytopenia: serious and under-reported? Our experience of 25 cases in 5 years

A. Y. N. Lim, K. Gaffney and D. G. I. Scott

Rheumatology Department, Norfolk and Norwich University Hospital, Norwich NR4 7UY, UK.

Correspondence to: A. Y. N. Lim. E-mail: anitalim{at}doctors.org.uk


    Abstract
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 Abstract
 Introduction
 Methods
 Results (Table 1)
 Discussion
 References
 
Objective. To ascertain the extent of methotrexate (MTX)-related pancytopenia at the Norfolk and Norwich University Hospital (NNUH) between 1999 and 2004.

Methods. Patients were identified by a department database search, review of pharmacy records and personal communication. Pancytopenia was defined as white blood cell count (WBC) <3.5 x 109/l, haemoglobin (Hb) <11 g/dl and platelet count <130 x 109/l. Severe pancytopenia was defined as WBC <2.0 x 109/l, Hb <10 g/dl and platelet count <50 x 109/l.

Results. Twenty-five patients had MTX-induced pancytopenia. Eleven patients were taking folic acid and one folinic acid. The median dose of MTX was 12.5 mg weekly (interquartile range 5.625 mg) and median duration of treatment 36 months (interquartile range 40.5 months). The severity of pancytopenia correlated with the dose (P = 0.04). The numbers of patients with potential risk factors were: renal insufficiency, 8; pre-existing folate deficiency, 7; age >75 yr, 15; hypoalbuminaemia, 18; pre-existing infection with hip prosthesis, 1; possible drug interactions, 18; dosing errors, 1; and polypharmacy, 15. Pancytopenia was detected by routine blood monitoring in nine patients. There were seven deaths (28% mortality), five from sepsis and two from acute myeloid leukaemia.

Conclusion. This is the largest reported individual case series of MTX-induced pancytopenia. With the increasing long-term use of MTX, it is important that patients be monitored for haematological side-effects as pancytopenia can be a late manifestation. Pharmacogenetics may hold the answer to predicting who is at risk of this potentially fatal complication of MTX.

KEY WORDS: Methotrexate, Pancytopenia, Hypoalbuminaemia, Nutritional status, Mucositis, Folic acid, Polymorphisms


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results (Table 1)
 Discussion
 References
 
Methotrexate (MTX) is the commonest disease-modifying anti-rheumatic drug (DMARD) used in the treatment of rheumatoid arthritis (RA) [1]. It is prescribed as monotherapy and in combination with other DMARDs and biological agents. In MTX-treated RA patients, the prevalence of haematological toxicity, including leucopenia, thrombocytopenia, megaloblastic anaemia and pancytopenia, is estimated to be 3% [2]. The extent of pancytopenia, a serious and unpredictable adverse effect of low-dose MTX, may be underestimated. Because of anecdotal experience of increasing hospital admissions due to MTX-related pancytopenia at the Norfolk and Norwich University Hospital (NNUH), a retrospective review of all relevant case notes was undertaken.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results (Table 1)
 Discussion
 References
 
Between 1999 and 2004, twenty-five patients with MTX-induced pancytopenia were identified by a department database search, review of pharmacy records and personal communication. Pancytopenia was defined as white blood cell count (WBC) <3.5 x 109/l, haemoglobin (Hb) <11 g/dl and platelet count <130 x 109/l. Severe pancytopenia was defined as WBC <2.0 x 109/l, Hb <10 g/dl and platelet count <50 x 109/l. Patient notes were reviewed and the following data recorded: age, sex, indication for MTX, median dose and duration of treatment, concomitant therapy, erythrocyte mean corpuscular volume (MCV), serum albumin, liver and renal function. The catchment population served by the NNUH is 550 000. The prevalence of RA, as estimated by the Norfolk Arthritis Register (NOAR), is 0.8%. Between 1999 and 2004, NOAR recorded 25% of RA patients receiving MTX. A substantial number of patients are treated with MTX for other inflammatory conditions. Therefore, the prevalence of MTX-induced pancytopenia in this cohort is estimated at approximately 1.9%.


    Results (Table 1)
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 Abstract
 Introduction
 Methods
 Results (Table 1)
 Discussion
 References
 
Patient demographics
The median age was 76 yr interquartile range (IQR) 13; 17 female and eight male. The indications for MTX were: RA (19), RA/SLE (1), Wegener's granulomatosis (WG) (3), psoriatic arthritis (1) and giant cell arteritis (1). Pancytopenia was detected in nine by routine blood monitoring according to standard British Society of Rheumatology guidelines. Seven presented with bleeding (gums, urinary tract, epistaxis, vaginal and rectal). Oral mucositis was documented in 10 at presentation; seven had severe pancytopenia. One presented with an acute coronary syndrome secondary to anaemia. Concomitant sepsis was identified in nine, and five subsequently died.


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TABLE 1. Patient data

 
Associations
Eight had renal impairment (mean serum creatinine 235 µmol/l). Six had an increased MCV; low red cell folate levels were recorded in seven. Hypoalbuminaemia (mean serum albumin 26 g/l) was documented in 18. Three had abnormal liver function tests [alanine transferase (ALT) levels more than twice the upper limit of normal]. Six had not had regular blood monitoring. Two had developed pancytopenia previously with MTX, necessitating discontinuation. The development of pancytopenia in one patient coincided with the change in dispensing practice from two 10-mg MTX tablets weekly to eight 2.5-mg tablets. She took 2.5 mg daily for 6 days and 5 mg on the seventh day. One was diabetic. The youngest (24 yr) had a past history of anorexia nervosa; a liver biopsy showed severe steatosis consistent with poor nutritional status.

Concomitant drugs
Fifteen were taking five or more concomitant drugs: coxibs (5), proton pump inhibitors (7), diuretics (11), angiotensin converting enzyme inhibitors (5), beta-blockers (3) and oral bisphosphonates (10). One developed pancytopenia within 4 days of receiving intravenous pamidronate. One patient with WG was receiving concurrent cotrimoxazole. This therapeutic combination has previously been highlighted as a causative factor in pancytopenia.

Dosing issues
The median dose of MTX was 12.5 mg weekly (IQR 5.625 mg) and median duration of treatment with MTX 36 months (IQR 40.5 months). Three were receiving weekly parenteral MTX. Eleven were taking folic acid (ranging from 5 mg weekly to 5 mg for 6 days, avoiding the day of MTX) and one was taking folinic acid 15 mg daily.

Severity of pancytopenia
These data are outlined in Table 2. Ten had severe pancytopenia; 50% died; four were taking folic acid. Of the 15 with non-severe pancytopenia, eight were taking folic acid. The nine patients who were detected by blood monitoring comprised eight with non-severe and one with severe pancytopenia; all recovered except one, who died of acute myeloid leukaemia (AML). Eighteen were hospitalized; nine required blood transfusion, 17 folinic acid rescue and the remaining eight daily folic acid. The mortality rate was 28%. Deaths were due to sepsis in five (all had severe pancytopenia) and AML in two (both had non-severe pancytopenia). Non-parametric testing showed no significant differences between the groups, either by dose or by duration of treatment. However, when comparing those who recovered from severe or non-severe pancytopenia, the former were taking a significantly higher dose (median 20 mg, IQR 9.375 mg) than the latter (median 10 mg, IQR 3.125); P = 0.04, Mann–Whitney U test. Although the duration of treatment may be a risk factor in the development of severe pancytopenia, this was not statistically significant.


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TABLE 2. Patient outcomes

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results (Table 1)
 Discussion
 References
 
Pancytopenia, a rare but potentially fatal complication of MTX therapy, may develop suddenly and without warning signs. It can occur early (within 1–2 months of starting MTX, independently of dose and route of administration), when it is rarely avoidable, possibly reflecting an idiosyncratic reaction. More commonly, however, it occurs late [3], as in our series, suggesting a cumulative effect.

MTX is a highly selective competitive inhibitor of the enzyme dihydrofolate reductase and consequently reduces the production of thymidylate and DNA synthesis. Tissues undergoing rapid cellular turnover with a high fraction of the cells in S phase cycle (oral mucosa, gastrointestinal tract, bone marrow cells and testicular tissue) are the most susceptible to its cytocidal effects. Folate deficiency and MTX toxicity share some similar symptomatology. Depleted intracellular folate levels have been documented in hepatocytes and peripheral blood lymphocytes of RA patients treated with MTX [4, 5]. Folate supplementation was postulated to be useful in reducing some of the toxic manifestations associated with low-dose MTX therapy when initial plasma homocysteine levels (an indicator of folate status), were found to be predictive of future toxicity during MTX therapy in folate-unsupplemented patients [6].

No consensus exists among specialists who use low-dose MTX as to the optimal dose and timing of folate supplementation. Although folic acid supplementation has been shown to reduce the likelihood of derangement of liver function tests [7] as well as mucosal and gastrointestinal side-effects [8], higher mean doses of MTX have had to be used for equivalent efficacy [9, 10]. As yet, no studies have proven an unequivocal protective effect of folate supplementation on MTX-related haematological toxicity. In our cohort, four patients with severe pancytopenia and eight patients with non-severe pancytopenia were taking folic/folinic acid supplements. Of the seven fatalities, four were taking folic acid (two had AML). The extent to which mucositis and gastrointestinal toxicity have been virtually eliminated by the addition of folic acid in patients who may still encounter occasional bone marrow suppression provides evidence of the distinctive susceptibility of these specific tissues to MTX. Two of our patients had previously developed pancytopenia with MTX which recurred when they were rechallenged despite folic acid supplementation.

Stomatitis has been shown to precede or accompany pancytopenia and should be a warning sign. Patients with mucositis and neutropenia have a relative risk of septicaemia that is greater than four times that of individuals without mucositis [11]. These ulcers act as sites of secondary infection and portals of entry of endogenous oral flora. In addition, the oral flora of neutropenic patients differs from that of the healthy population as it is rich in Gram-negative organisms and typical {alpha}-haemolytic streptococci [12]. Seventy per cent of our cohort with severe pancytopenia had documented oral mucositis.

Why does pancytopenia occur? The reasons are multifactorial. MTX and folates compete at various stages, especially cellular uptake, cellular storage as polyglutamates and binding to enzymes, because of their structural similarity. The ability of MTX to undergo polyglutamation, which alters the spectrum of enzymes inhibited by the drug, is one of several properties that underlie cytotoxicity. MTX is polyglutamated and retained for a long period in the liver of RA patients [4], as well as bone marrow myeloid precursors [13] and human fibroblasts [14]. The accumulation of MTX polyglutamates in the liver reduces the polyglutamation of natural folates in that tissue [15] and may account for the chronic hepatotoxicity associated with MTX.

Dietary and genetic factors influence the disposition of cellular folate. Poor nutritional status has been associated with increased risk of toxicity from MTX [16, 17] because of an approximate two-fold decrease in clearance of MTX. The youngest patient had hepatic steatosis due to poor nutritional intake—an under-recognized risk factor for MTX toxicity. Exogenously administered reduced folates reverse MTX toxicity in a competitive manner.

Advanced age is a significant predictor for termination of MTX due to toxicity [18]. Fifteen of our 25 patients were over the age of 75; age-related decline in renal tubular function, not reflected in the face of normal renal function, may have contributed to MTX-associated pancytopenia. MTX is filtered by the glomeruli and undergoes secretion and reabsorption within the tubule. Reabsorption can occasionally be saturated earlier, resulting in increased renal clearance [19]. Conversely, patients with impaired renal function may have reduced drug clearance and be at risk of developing toxicity [20]. Delayed drug clearance in the elderly is possibly due to prolonged enterohepatic circulation, hence placing them at higher risk of pancytopenia. The elderly are more likely to have poor nutritional status. Age is also a determinant of whether patients will survive pancytopenia and its associated complications, such as sepsis, as the median age of our fatalities was 77 yr whereas the median age of those who recovered from severe pancytopenia was 61 yr.

Hypoalbuminaemia in RA may be due to increased albumin turnover, presumably caused by high consumption of albumin at sites of inflammation and poor nutritional status. Occult chronic liver disease may also be reflected in low serum albumin. The mean protein binding of MTX to serum albumin is 42–57%, whereas the metabolite (7-hydroxymethotrexate) is extensively (91–93%) bound. Significant interindividual variations in the activity of binding proteins contribute greatly to the efficacy and potential toxicity of MTX as this influences the influx of MTX into cells and its rate of clearance by the kidneys. Hypoalbuminaemia results in increased levels of free MTX because MTX binding to serum albumin is proportional to the amount of albumin, resulting in increased risk of myelotoxicity [21].

The unusual weekly dosing schedule of MTX has been emphasized by the National Patient Safety Agency, an organization that monitors adverse events occurring in NHS-funded care and makes recommendations to reduce risk, as this can lead to dose error. Dividing the dose of MTX, which one patient did by taking 2.5 mg daily for 6 days and 5 mg on the seventh day, adds greatly to the toxicity of MTX. The toxic effect of MTX on normal tissues is more a function of duration of exposure to suprathreshold concentrations of drug rather than the peak level achieved [22]. The situation with drug monitoring is such that MTX needs to be monitored in the long term even when the patient has been well on a stable dose for many months. The importance of routine blood monitoring cannot be highlighted enough as nine of our 25 patients were detected in this manner; conversely, six patients in residential/nursing home care had not had blood monitoring, which may have identified them earlier. There is a strong argument for a nationwide DMARD database monitoring system to standardize monitoring protocols, thus enabling earlier detection of abnormal results and lesser morbidity.

Common single-nucleotide polymorphisms of several genes coding for folate-dependent enzymes can modulate the risk of adult acute lymphoblastic leukaemia (ALL) [28] and other conditions. The level of risk or protection with some single-gene nucleotide polymorphisms is further influenced by a person's nutritional folate status. The C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene has been identified as a risk factor for elevated ALT values in MTX treatment in RA patients [23]. A study of the effect of the C677T polymorphism on the toxicity of MTX in patients undergoing bone marrow transplantation showed that homozygous patients had higher toxicity, as evidenced by increased oral mucositis and delayed platelet recovery [24]. Toffoli et al. reported that neutropenia following MTX chemotherapy was more common in patients with the 677TT polymorphism [25]. Berkun and colleagues have shown that the 1298CC polymorphism of the MTHFR gene protects against MTX-related adverse effects in RA patients, while the 1298AA genotype is associated with such adverse effects despite higher doses of folate supplementation [26].

Skibola et al. have shown that individuals with the C677T MTHFR TT genotype and both homozygous recessive and heterozygous A1298C MTHFR genotypes have a decreased risk of acute lymphoblastic leukaemia, indicating that folate depletion may play a role in the development of this malignancy [28]. Kolte et al. have described four cases of AML occurring in the setting of low-dose weekly MTX therapy for RA. Although they concluded that this was coincidental [27], we also identified two patients with AML. The mechanistic link between the C677T polymorphism (and possibly other single-nucleotide polymorphisms) and folate nutritional status may involve chromosome breakage and DNA methylation, factors implicated in the development of ALL [28]. It is possible that MTX contributed to folate depletion and, in the presence of single-nucleotide polymorphisms, resulted in AML in our two patients.

The permutation of folate nutritional status, common allelic variation in genes coding for folate-dependent enzymes, altered gene expression and impaired nucleic acid elaboration linked to folate metabolism possibly modulates the risk of developing pancytopenia secondary to MTX. More attention should be paid to patients’ nutritional status before commencing MTX; we advocate caution in the elderly whose serum albumin is below 28 g/dl and those with renal impairment. Folic acid supplementation at 5 mg weekly should be considered in all patients taking MTX.

In our experience, MTX-induced pancytopenia is more common than expected and is probably under-reported. We recommend vigilance for this late and potentially fatal complication of MTX therapy.

D.G.I.S. has received support from pharmaceutical companies involved in biological agents (Wyeth, Schering Plough, Abbott) which are often given in combination with methotrexate. The other authors have declared no conflicts of interest.


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 Abstract
 Introduction
 Methods
 Results (Table 1)
 Discussion
 References
 

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Submitted 17 January 2005; revised version accepted 12 April 2005.