THE TREATMENT OF schizophrenia with clozapine is known to induce granulocytopenia in 1.5% to 2.0% and severe agranulocytosis in 0.05% to 2% of patients.1-3 This side effect has severely limited the use of clozapine as a first-line medication, despite the fact that clozapine has been found to be superior to classic antipsychotic agents in treating therapy-resistant schizophrenia.4, 5
A growing body of evidence indicates that delays in providing effective treatment for patients with psychosis have a negative effect on the outcome.6-8 Moreover, treatment refractoriness, i.e., the resistance of positive symptoms to neuroleptics, is a major cause of prolonged and persistent disability in psychosis.9
Thus far, clozapine has proven to be the most effective therapeutic alternative for patients with schizophrenia that is refractory to conventional neuroleptics; 30% of such patients benefit from it.4 Compared with conventional antipsychotic treatment, clozapine has been shown to be effective in early-onset schizophrenia,10 which has been associated with neuroleptic resistance.11 Moreover, clozapine treatment has been shown to significantly improve several domains of cognitive functions compared with classic antipsychotic drugs, thus contributing to the reduction of disability in schizophrenia.12-14
There have been efforts to define predictors of response to clozapine, as well as risk factors for agranulocytosis, to identify those patients who benefit therapeutically and have a minimal risk of bone marrow suppression.1, 15, 16 Several reports indicate that clozapine-induced agranulocytosis is associated with specific human leukocyte antigen (HLA) haplotypes.17-19 However, biological predictors of the effects of clozapine treatment are not yet available in clinical practice.
In our previous study, we discovered a strong association between HLA-A1 and a good therapeutic response to clozapine in patients refractory to classic antipsychotic drugs.20 In the present extended study, we examined whether these favorably responding patients carrying the HLA-A1 allele were at risk of developing agranulocytosis.
The subjects of this study were Finnish-born hospital patients and outpatients meeting diagnostic criteria for a schizophrenic disorder according to DSM-III-R. All of the patients were receiving clozapine treatment. The patients were divided into two groups on the basis of their response to clozapine. One group, called "clozapine responders," comprised 19 patients (8 women, 11 men; mean age, 35.7 years [SD = 9.2]) with a clinical response to clozapine and a lack of response to classic antipsychotics, based on the operational criteria for treatment refractoriness, which we used previously.20 In this group, the mean dose of clozapine was 494 mg (SD = 160). The second group consisted of 26 patients (15 women, 11 men; mean age, 34.9 years [SD = 12.5]) with a history of nonfatal clozapine-associated granulocytopenia (neutrophil granulocytes <1.5 × 109/L) or agranulocytosis (neutrophil granulocytes <0.5 × 109/L). The treatment lasted 161.4 days (SD = 342.5), and the mean dose of clozapine was 405.8 mg (SD = 153.2). Healthy Finnish blood donors (N = 120) served as controls. The study protocol was approved by the ethical committee of the hospital. Four subjects belonged to multiply affected families that were collected in a large genetic study approved by the ethics committee of the National Public Health Institute.21, 22 The subjects gave their informed consent after the procedure had been fully described to them.
HLA typing procedures
HLA-A and -B typing was performed using fresh peripheral blood T cells by antibody-mediated lymphocytotoxicity using commercial typing trays (2 × 72 well trays) (Pel-Freez; Brown Deer, WI). In the agranulocytosis/granulocytopenia group, four patients were typed only for the A locus and at the DNA level, using an HLA-A "low resolution" PCR-SSP typing kit (Dynal, Oslo, Norway).
The statistical significance was tested using the Fisher exact test. p Corrected values were corrected with the number of comparisons made (N = 23). p Values lower than 0.002 were considered significant at the corrected level, and the odds ratios (OR) are quoted.23
Table 1 shows the HLA antigen frequencies in patients with granulocytopenia or agranulocytosis, as well as in clozapine responders. In patients 1 to 4, only the A locus was analyzed. The frequency of HLA-A1 was 11.5% in patients with clozapine-induced agranulocytosis or granulocytopenia, whereas the frequency of HLA-A1 was 57.9% (p = 0.0013) in the group refractory to conventional antipsychotics but responsive to clozapine. HLA-A1 occurred at a frequency of 20.1% in the healthy Finnish controls. The difference in HLA-A1 frequencies between the agranulocytosis/granulocytopenia group and the responder group was statistically significant. In addition, there was an association between agranulocytosis or granulocytopenia and the A28 specificity. The frequency of A28 was 30.7% in the patients with agranulocytosis or granulocytopenia, compared with 5.3% in the responder group (p = 0.027). After correction, however, the p value was no longer significant. Moreover, the frequency of the B16 allele was 27.3% in the agranulocytosis/granulocytopenia group, whereas it was only 9.3% among the healthy controls (p = 0.027). Similarly, HLA-B40 was expressed in nearly 23% of the patients who developed agranulocytosis or granulocytopenia but in none of the clozapine responders. Again, after correction, the p value was not significant. The frequency of the B16 allele did not differ significantly between the two study groups.
Table 2 shows the neutrophil count and the presence of HLA class I antigens in the patients. According to the type of neutropenia, they could be classified into a mild-to-moderate type (absolute neutrophil count of 0.5-1.5 × 109) and a more severe type (absolute neutrophil count <0.5 × 109). Of the 26 patients, 8 had severe neutropenia (6 women and 2 men). The mean age in the entire group was 34.9 years (SD = 12.5), but it was 43.0 years (SD = 10.6) in the group with severe neutropenia. These data are consistent with previous reports associating increasing age and female gender with an increased risk of clozapine-induced agranulocytosis.1 The groups did not differ regarding HLA-A28 expression. None of the patients with severe neutropenia carried the HLA-A1 allele, whereas 16% of the patients with mild-to-moderate neutropenia expressed this allele.
We have earlier reported that patients with schizophrenia whose disease is refractory to conventional antipsychotic treatment but responsive to clozapine have a strong association to HLA-A1. Nearly 58% of these patients expressed this allele.20 On the basis of this finding, HLA-A1 is a marker for patients who benefit from clozapine treatment. However, there is a risk of granulocytopenia related to this treatment. Therefore, we wanted to determine whether patients developing granulocytopenia/agranulocytosis show the same association or not. Our results suggest that HLA-A1 not only predicts a good therapeutic outcome with clozapine, but also a relatively low risk of agranulocytosis. It is interesting to note that none of the patients developing severe neutropenia expressed this allele.
Previous studies of the HLA association in clozapine-induced agranulocytosis indicate that a high risk for agranulocytosis may correlate with certain HLA haplotypes. Associations with HLA-B16, B38, DR4, DR2, and DQ1 have been reported.17-19, 24
In our study, albeit with a relatively small number of patients, we found a weak association with HLA-B16 and agranulocytosis or granulocytopenia. This is in keeping with findings from earlier studies. An association between HLA-B16 and clozapine-induced agranulocytosis has been reported in the Ashkenazi Jewish population, as well as in a native American patient.18,25 Claas and colleagues26 analyzed 103 patients with clozapine-associated granulocytopenia or agranulocytosis, 65 of whom were Finnish. Although they did not find any significant association between the presence of a particular HLA antigen and susceptibility to clozapine-induced granulocytopenia or agranulocytosis, the frequency of HLA-B16 was elevated in patients with bone marrow suppression. In addition to B16, our study shows a new association to A28, which gives a higher odds ratio than for B16 (OR of 8.0 vs. 3.2, respectively).
Associations with HLA class II specificities suggest the involvement of an immune-mediated mechanism of the agranulocytosis. Association with an HLA class-I antigen might rather indicate that it serves as a marker for responsible gene(s) within the HLA area on chromosome 6. This is in accord with earlier reports of associations between certain major histocompatibility (MHC) antigens and individual susceptibility in which HLA alleles have been suggested to be in linkage disequilibrium with other genes in the MHC region.17, 27, 28
A link between a specific HLA and agranulocytosis, together with several other findings, may speak in favor of an immunomediated mechanism. In the serum of patients with clozapine-induced agranulocytosis, complement-mediated toxicity to neutrophils has been shown, and it was attenuated by IgM antibodies.29 Clozapine treatment has been reported to increase dose-dependently concentrations of soluble interleukin-2 receptors in peripheral blood.30 A metabolite of clozapine, norclozapine, suppressed in vitro antigen-stimulated proliferation of isolated human lymphocytes. Also, clozapine dose-dependently suppressed the granulocyte-macrophage colony-stimulating factor and neopterin release from human blood mononuclear cells in vitro. 31, 32 However, there is no direct evidence of the presence of antidrug antibodies, antineutrophil antibodies, or antimyeloid antibodies.33, 34
Our earlier findings suggested that HLA-A1 could be in linkage disequilibrium with genes predisposing to schizophrenia on chromosome 6, and this would help define a subgroup of patients with schizophrenia.20 Our present findings indicate that HLA-A1-positive patients, who are resistant to conventional neuroleptics, have a relatively low risk of agranulocytosis and a good therapeutic outcome with clozapine therapy. We therefore suggest that HLA typing may aid in the selection of patients for clozapine medication at an early stage of the disease, especially among patients with early-onset schizophrenia. Indeed, early and effective antipsychotic treatment could prevent some of the deterioration that many patients currently experience. However, more studies are needed with larger numbers of patients having a different background than the genetically homogeneous Finnish population to confirm these findings.
The authors gratefully acknowledge the contribution of the Orion Company, Espoo, Finland, Novo Nordisk Foundation, and the Wilhelm and Else Stockmann Foundation, Finland.
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