1 Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow Klinikum, Berlin, Germany and 2 Service d'Hématologie Biologique, Hôpital Hôtel-Dieu, Paris, France
Keywords: anti-erythropoietin antibodies; epoetin treatment; loss of efficacy; pure red-cell aplasia
Biopharmaceuticals generated by expression of DNA in cell lines have great potential for the treatment of diseases, in which the endogenous production of a specific protein is inadequately low and/or its administration has beneficial modulating effects on disease processes. Although these drugs are designed as copies of endogenous molecules, immunogenicity is a recognized risk. Factors that have been associated with an immunogenic potential include subtle differences in protein structure and in glycosylation (which is inherently variable), contaminants of the production process, the formulation, storage conditions, patient associated variables and the mode of application, with the i.v. route usually carrying the lowest risk [1].
Erythropoietin (Epo) produced by recombinant DNA technology (epoetin) is probably the most successful biopharmaceutical to date. It was introduced for the treatment of renal anaemia in the late 1980s as an i.v. drug. A few years later when first attempts were made to administer it s.c. at least some investigators were concerned about an increased risk of antibody formation. However, the s.c. application proved to be feasible and safe. Due to a lower dose requirement it became the preferred route of administration in many countries. Epoetin-induced antibodies remained a very rare complication for many years. Recently, however, a significant increase in the frequency of this complication, which leads to severe inhibition of red cell production, was noted.
Prevalence of epoetin-induced PRCA
During the first 10 years of therapy, three cases of epoetin-associated pure red-cell aplasia (PRCA) were published [24]. In addition, in one patient, who never received the recombinant hormone, autoantibodies against Epo were demonstrated as a cause of PRCA [5]. Since 1998, a significant increase has been noted in the number of patients developing severe anaemia during the course of epoetin treatment due to neutralizing antibodies. A detailed description of a series of 13 cases was published about 1 year ago [6]. Meanwhile, about 200 suspected cases in patients with chronic kidney disease have been reported to health authorities. In about 120 of these cases antibodies against Epo have been demonstrated.
Definition and diagnosis of PRCA
PRCA is a progressively developing, severe, isolated anaemia, with sudden onset and almost complete absence of red cell precursors from an otherwise normal bone marrow [7]. As a reflection of almost complete cessation of red cell production, the blood reticulocyte count is very low (<10 000/mm3). The haemoglobin concentration declines at a rate of 0.1 g/dl/day (slightly less than 1 g/dl/week), corresponding to a red cell life span. In order to avoid very severe anaemia and maintain an acceptable haemoglobin level, transfusion of
1 U of red blood cells per week is required.
Among many possible causes of PRCA, the most important ones are listed in Table 1. The diagnosis of epoetin-induced PRCA should be based on the clinical presentation, the absence of red cell precursors in the bone marrow and the presence of anti-Epo antibodies (Table 2
). A bone marrow aspirate is sufficient for this purpose and a biopsy is not required, but the investigator needs special experience in making the diagnosis. Radioimmunoprecipitation reactions and ELISA-based assays have been developed for detection of anti-Epo antibodies. Care has to be taken to avoid unspecific binding and thus false-positive results, in particular when ELISAs are being used. A new ELISA system has been developed to minimize these risks, in which antibodies are only detected if their two arms bind sequentially to two Epo molecules (A. Haselbeck, F. Hoffmann-La Roche, personal communication). In vitro bioassays based on incubation of red cell precursors or a cell line, which depends on Epo, with patient serum are the only available tests to demonstrate and quantify the neutralizing activity of antibodies against Epo [6]. So far, in patients diagnosed to have PRCA, all antibodies binding in the radioimmunoprecipitation reaction were also found to be neutralizing. Ongoing studies are investigating whether anti-Epo antibodies may contribute to Epo resistance in patients who do not present with the classical picture of PRCA. To date, however, there is no evidence for this hypothesis [8]. The time interval between onset of epoetin therapy and loss of efficacy in patients demonstrated to have epoetin-induced PRCA is at least 3 weeks and averages
9 months (Figure 1
).
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Causes of epoetin-induced PRCA
Given the severity of the anaemia associated with PRCA, it is unlikely that this complication was overlooked in many patients during the first 10 years of epoetin therapy. There rather appears to be a true rise in incidence, although awareness has certainly also increased during recent months. One of the factors that is clearly associated with the development of anti-Epo antibodies is its s.c. application. Very few patients, if any, treated by the i.v. route only have developed this complication. Skin reactions at the site of recent or former s.c. injections have rarely been observed and illustrate the importance of local immune reactions in the subcutis [9]. In addition, there seem to be differences among different brands of epoetin. The majority of cases reported are associated with the use of epoetin alfa distributed outside the US, which is manufactured at a different site than the preparation distributed within the US [10]. Nevertheless, four cases of PRCA have also been observed with epoetin alfa in the US (B. Maroni, Amgen, personal communication, January 2003). The report by Casadevall et al. [6] also included one patient who developed PRCA under exclusive treatment with epoetin beta, and Krüger et al. [11] in this issue of NDT report a second epoetin beta case. The company producing epoetin beta states that as of December 2002 they have received five reports of patients treated exclusively with epoetin beta in whom PRCA was diagnosed and who tested positive for anti-erythropoietin antibodies and reports of three additional cases, in which an association between the use of epoetin beta and the development of PRCA cannot be excluded (E. Fröhlich, F. Hoffmann-La Roche, personal communication, March 2003). There have also been rare reports of allergic reactions including skin rash and urticaria associated with darbepoetin alfa, a hyperglycosylated epoetin molecule. To date, however, there have been no reported cases of antibody-mediated PRCA caused by darbepoetin alfa (B. Maroni, Amgen, personal communication, January 2003). Naturally, in patients who have received more than one epoetin preparation the proof of causality between a particular brand and the development of antibodies can be difficult.
Thus, although a low incidence of PRCA has been found with different epoetins, a higher risk of immunogenicity appears to be associated with one preparation. The increase in reported cases coincides with the removal of human serum albumin (HSA) from the ex-US formulation of epoetin alfa, in order to comply with new regulations from the European regulatory authorities. Polysorbate 80 and glycine were added instead of HSA. It has been hypothesized that this formulation is less stable, so that under adverse circumstances, such as prolonged exposure to increased temperatures, aggregates of Epo molecules form, which increase the likelihood of antibody formation following s.c. administration. It has also been considered that small amounts of silicone, used to lubricate pre-filled syringes, may enhance the antigenicity. The formulation of epoetin beta has always been HSA-free, but the stabilizer composition differs from that of epoetin alfa. No comparison of the stability of different preparations has been published. Subtle differences exist between the carbohydrate moieties of epoetin alfa and beta [12,13], but whether these have any impact on immunogenicity remains unknown. As far as investigated, the anti-Epo antibodies in patients with PRCA are directed against the protein moiety of the molecule [6].
Interestingly, cases of epoetin-induced PRCA have so far not been observed in cancer patients, although the anaemia of malignant disease has become a frequent indication for epoetin therapy. Potential reasons include a shorter duration of therapy, reduced life expectancy and unspecific immunosuppression. Due to the many possible reasons for a worsening of anaemia in advanced cancer, the diagnosis might also be more easily missed than in patients with renal disease. Very recently, anti-Epo antibodies and PRCA were diagnosed in two patients with myelodysplastic syndrome, who received epoetin therapy for 7 and 16 months, respectively (N. Casadevall, S. Giraudier, L. Quint, unpublished data). One was treated with epoetin alfa, the other with epoetin beta.
Therapy
When epoetin-induced PRCA is suspected, treatment with epoetin needs to be stopped. Patients do not respond to an increase in dose. It is important that the patient is not switched to another recombinant erythropoietic protein (epoetin alfa, beta or darbepoetin alfa), since all antibodies detected so far are cross-reactive with the endogenous and all recombinant Epo molecules [6,9]. Continuation of epoetin therapy in the presence of anti-Epo antibodies can lead to anaphylactoid reactions [9]. Different immunosuppressive regimens have been tried, including corticosteroids, cyclophosphamide, high dose intravenous immunoglobulins and plasmapheresis, and were found to be associated with variable success. Cessation of epoetin therapy alone without immunosuppressive therapy has so far only rarely been found to reverse antibody formation and improve erythropoiesis. Transplantation seems to provide an immediate cure presumably due to the effects of immunosuppressive therapy, and despite the theoretical potential of graft-derived Epo to boost the immune response (J. Rossert, personal communication, January 2003).
Preventive measures
The manufacturer of the ex-US brand of epoetin alfa associated with the recent increase in reports of PRCA states that efforts were undertaken to enhance manufacturing controls, reduce product exposure to ambient temperatures, and reduce the quantity and variability of silicone in syringes (http://www.jnj.com/news/jnj_news/20020917_142708.htm). In July 2002, a recommendation was issued to prefer i.v. application of epoetin alfa. More recently, s.c. use of epoetin alfa has become a contraindication in patients with chronic kidney disease (http://www.jnj.com/news/jnj_news/20021202_143150.htm). Manufacturers of epoetin beta and darbepoetin alfa have not been required by regulatory authorities to change the indications of their products, but warnings have been added to the prescription information of these drugs.
Regular monitoring of the reticulocyte count performed by automated flow cytometry has been recommended as an integral part of epoetin therapy in order to allow early detection of impaired erythropoiesis due to anti-Epo antibodies [14]. It is predictable that a fall in reticulocytes preceeds the development of severe anaemia, yet the clinical benefit of an earlier detection is undetermined. Irrespective of whether reticulocytes are routinely monitored, the reticulocyte count should certainly be one of the first steps during work-up of cases in which loss of efficacy of epoetin is observed.
Screening tests for anti-Epo antibodies are required to obtain more information about their incidence, prevalence and clinical significance (see above). However, such assays are only recommended in the context of controlled scientific investigations with appropriate validation of the assay technique. Otherwise, we do not consider screening tests to be useful in patients, in whom there is no clinical evidence for PRCA including a low reticulocyte count.
Conclusions
Despite understandable concern about an increased number of cases, PRCA due to anti-Epo antibodies remains a very rare complication, that is not per se fatal and principally reversible. Although it is difficult to predict future developments, at present there does not seem to be an exorbitant increase in incidence. It still appears true that the ratio of risk to benefit of epoetin is matched by few other pharmacologic agents [15].
Nevertheless, physicians using the drug must be aware of signs and symptoms of this complication and the appropriate diagnostic procedures. It has also become apparent that adherence to handling instructions is probably more important than previously recognized.
At present, it appears unlikely that one single cause explaining the increase in incidence will ever be identified. A follow-up of each individual case is certainly desirable to identify factors associated with the occurrence of this complication and to collect further information on the natural progression and response to therapeutic intervention. It is also obvious that the potential risk for induction of neutralizing antibodies needs to be considered for all second generation molecules and generic preparations of recombinant Epo.
Notes
Correspondence and offprint requests to: Prof. Kai-Uwe Eckardt, Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany. Email: kai-uwe.eckardt{at}charite.de
References