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Gene transfer of the cytidine deaminase (CDD) cDNA has recently been shown to induce cellular resistance to cytarabine (AraC) in vitro. To investigate the role for CDD in acute myeloid leukaemia (AML) we analysed the CDD activity and CDD gene structure in blast material from well-defined patients with untreated and AraC refractory (RF) AML. Median CDD activity in previously untreated AML was significantly lower than in RF-AML blasts (P = 0.015) and was significantly lower in patients with complete remission than with blast persistence following induction chemotherapy (P = 0.043). Structural investigation of the CDD gene by Southern analyses and RT-PCR showed no detectable aberrations. Sequence analysis of the CDD cDNA from nine RF-AML patients showed inconsistent aberrations in three patients. Semiquantitative assessment of CDD mRNA expression revealed a significant correlation with CDD activity. In conclusion, concordant with another recent study our data suggest a correlation of pretherapeutic CDD activity with induction treatment response. Besides the previously described prognostic impact of mdr1 expression, this result could be useful for the development of risk-adapted AML treatment strategies and warrants further studies of CDD activity in well-defined cohorts of AML patients and of the mechanisms involved in the regulation of CDD activity.
Cytostatic induction chemotherapy of adult acute myeloid leukaemia (AML) with daunorubicin or idarubicin plus cytarabine (AraC) results in complete remission rates of 60–80%. In certain subgroups of patients long-term disease-free survival (DFS) rates of around 40–60% are achieved with myeloablative regimens followed by allogeneic haemopoietic progenitor cell transplantation (alloPCT) or with intensive post-remission regimens including high-dose AraC (HD-AraC) ( Mayer et al, 1994 ). In unselected cohorts of AML patients, however, DFS rates are in the range of 20–25%. Thus, cytostatic drug resistance towards anthracyclines and AraC remains the major obstacle to long-term DFS in the majority of AML patients.
Further improvement of AML therapy is conceivable with the development of risk-adapted treatment strategies based on pretherapeutic individual or tumour biological determinants. Prognostic factors which have been previously established are age at diagnosis and karyotypic aberrations, and may possibly include antecedent haematological disorders, LDH levels, and autonomous growth of leukaemic cells in vitro ( Mrózek et al, 1997 ; Mayer et al, 1994 ; Büchner et al, 1993 ; Hunter et al, 1993 ). Theoretically, among the most sensitive predictors for treatment response should be those associated with mechanisms of cellular resistance towards anthracyclines and AraC. In fact, although not yet firmly established, there is accumulating recent evidence that the expression of an mdr1 phenotype in leukaemic blasts correlates with anthracycline resistance and is an adverse prognostic factor for reaching a complete remission and long-term DFS ( Schröder et al, 1996a ; Campos et al, 1992 ; Nuessler et al, 1997 ; Del Poeta et al, 1996 ; van den Heuvel-Eibrink et al, 1997 ; Kasimir-Bauer et al, 1998 ). In contrast, although several putative in vitro mechanisms of cellular AraC resistance have been identified during the last two decades, controversy exists as to their potential clinical impact. Among others, these include an inactivation of deoxycytidine kinase (DCK; EC 126.96.36.199) and an increase of cytidine deaminase (CDD; EC 188.8.131.52) activity ( Capizzi et al, 1991 ).
Circumstantial evidence for a possible correlation between CDD activity and AraC resistance has been suggested by previous in vitro and in vivo studies ( Yusa et al, 1992 ; Honma et al, 1991 ; Riva et al, 1992 ; Kreis et al, 1977 ; Steuart & Burke, 1971; Colly et al, 1987 ; Jahns-Streubel et al, 1997 ; Tattersall et al, 1974 ). A causal relationship between these variables in vitro, however, has only recently been established by us and other groups ( Schröder et al, 1996b ; Neff & Blau, 1996; Momparler et al, 1996 ; Flasshove et al, 1997 ). In addition, we showed that recombinant proteins corresponding to two natural variants of the CDD cDNA which differ by a single amino acid are associated with significantly different deamination rates of AraC in vitro ( Kirch et al, 1998 ). As these data may suggest a structure–function relationship that could be relevant to clinical AraC resistance, we performed a structural analysis of the CDD gene in blasts from patients with newly diagnosed and recurrent AML. Considering the inconsistent data obtained from previous in vivo studies investigating the potential correlation of CDD or DCK activities with clinical AraC resistance, we also assessed the CDD and DCK enzymatic activities in well-defined subsets of AML patients with either sensitive, persistent or recurrent disease.
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- PATIENTS AND METHODS
Among the various mechanisms involved in cellular AraC resistance, several lines of evidence have indicated a role for DCK deficiency and/or CDD increase in its pathogenesis in vitro ( Meyers & Kreis, 1978; Bhalla et al, 1984 ; Richel et al, 1990 ; Kees et al, 1989 ; Rustum & Preisler, 1979; Owens et al, 1992 ; Yusa et al, 1992 ; Honma et al, 1991 ; Riva et al, 1992 ; Kreis et al, 1977 ). Corresponding clinical studies, however, yielded inconsistent results ( Steuart & Burke, 1971; Colly et al, 1987 ; Jahn-Streubel et al, 1997 ; Tattersall et al, 1974 ; Mejer & Nygaard, 1978; Smyth et al, 1976 ), which at least in part may be explained by differences in treatment schedules, AraC doses and response criteria, variations in enzyme assays, and the inclusion in several studies of patients' samples with blast percentages as low as 30–50% possibly confounding enzyme activities with those of normal haemopoietic cells ( Ho, 1973). Further evaluation of the putative role of these enzymes in the pathogenesis of AraC resistance has only recently become possible with the molecular cloning of their corresponding cDNAs ( Chottiner et al, 1991 ; Kühn et al, 1993 ; Bertling et al, 1993 ; Laliberte & Momparler, 1994). In fact, gene transfer studies clearly established a causal relationship between enzyme activity and cellular sensitivity (DCK) or resistance (CDD) to AraC in vitro ( Manome et al, 1996 ; Schröder et al, 1996b ; Neff et al, 1996 ; Momparler et al, 1996 ; Flasshove et al, 1997 ). Furthermore, structural analyses showed inactivating mutations of the DCK gene as a cause of DCK deficiency in vitro and revealed polymorphism at codon 27 of the CDD cDNA to correlate with significantly different deamination rates of AraC in vitro ( Owens et al, 1992 ; Stegmann et al, 1995 ; Kirch et al, 1998 ). So far, a molecular analysis of these genes in primary human AML blasts has been reported only for the DCK gene. In a study of AraC-resistant AML patients we previously showed that DCK cDNA mutations occur only rarely in vivo and therefore may not constitute a major mechanism of clinical AraC resistance ( Flasshove et al, 1994 ).
In the present study we first assessed DCK enzyme activities in patients with untreated, responsive and refractory disease and found no significant differences between these groups of patients. Although this result contrasts with data from some anecdotal studies, it is in accordance with another recent investigation ( Jahns-Streubel et al, 1997 ). Thus, as both our structural and functional analyses failed to indicate a role of DCK for AraC resistance in vivo, we next addressed the putative role of the CDD in cellular AraC resistance.
Possibly influenced by the methodological reasons cited above, previous studies of CDD activity in primary human AML blasts had yielded inconsistent results, either showing a significant correlation of treatment failure with (a) increased CDD activity ( Steuart & Burke, 1971), (b) high CDD or low DCK activity ( Colly et al, 1987 ), (c) a non-significant trend for increased CDD activity ( Tattersall et al, 1974 ), or (d) no such relationship ( Mejer & Nygaard, 1978; Smyth et al, 1976 ). In the present investigation of uniformly treated AML patients we observed a significantly higher median CDD activity in recurrent AML than in previously untreated patients and also found a significant correlation of median CDD activity with induction treatment response. Interestingly, these results are concordant to those of another recent study of uniformly treated, well-defined AML patients describing a significant correlation of CDD activity with early blast cell clearance ( Jahns-Streubel et al, 1997 ). Whereas the latter investigation also suggested a correlation of CDD activity with remission duration, this relationship, however, failed statistical significance in our analysis.
To elucidate the mechanism(s) associated with increased CDD activity, we performed Southern analyses in patients with refractory AML and found no indication for gene amplification or other genomic aberrations. Likewise, RT-PCR analyses covering the CDD ORF did not reveal aberrations in transcript sizes between patients with untreated, responsive or refractory disease. We next investigated the possible occurrence of nucleotide alterations within the CDD cDNA gene in AraC refractory patients. This analysis was based on our previous finding of the functional relevance of a single nucleotide exchange at codon 27 of the CDD cDNA gene ( Kirch et al, 1998 ). In addition, Stegmann et al (1995 ) had shown that AraC exposure of cells in vitro may lead to random mutations of the DCK gene with subsequent induction of AraC-resistant cell clones. As this, in analogy, might suggest the possible induction and selection for activating mutations within the CDD gene, we sequenced the CDD ORF in nine AML patients with recurrent HD-AraC-resistant AML. The internal deletion detected in one of three clones from one of these patients had no corresponding detectable amplification product in the RT-PCR analysis, thus questioning its functional relevance. Alignment of the unique amino acid mutations observed in part of the clones from the other two patients with the well-characterized E. coli CDD suggests that these mutations are located outside of functionally important domains ( Betts et al, 1994 ). Hence, whereas we have not yet determined the enzymatic activities of these mutated clones and no detailed structure–function analysis of human CDD is presently available, the functional impact of these mutations may be questionable.
Since structural aberrations of the CDD cDNA gene did not seem to represent a major cause of the observed differences of CDD activities between AraC-sensitive and AraC-resistant patients, we performed a semiquantitative assessment of CDD mRNA expression and found a significant correlation of signal intensities for PCR amplification products with CDD enzyme activities. This result is in accordance with recent preliminary data from another group ( Jahns-Streubel et al, 1997 ) and suggests that CDD activity is at least partly regulated at the transcriptional level. Because in vitro incubation of AML cells with the hypomethylating agent 5′-aza-2′-deoxycytidine was reported to lead to increased CDD expression ( Laliberte & Momparler, 1994; Momparler & Laliberte, 1990), we also analysed the methylation status of the CDD structural gene but found no differences in the patterns of restriction fragments between the different groups of patients. Since the regulatory region of the CDD gene has not been cloned, the latter experiments, however, do not yet allow conclusions on the potential impact of promoter methylation on the regulation of CDD mRNA expression.
In conclusion, consistent with data from several in vitro analyses and another recent clinical investigation of uniformly treated, well-defined AML patients the current study sheds further light on the possible relationship of CDD activity with AraC resistance in vivo. As to the establishment of risk-adapted strategies for AML therapy it may suggest further evaluation of the correlation of pretherapeutic CDD activity with early treatment response. Considering the high failure rate and the significant toxicity associated with HD-AraC consolidation therapy, it may as well warrant an evaluation of the relationship of pretherapeutic CDD activity with treatment outcome following HD-AraC containing post-remission therapy. To our knowledge, it represents the first structural analysis of the CDD gene in AML patients and indicates that CDD activity in vivo is correlated with transcriptional regulation rather than with CDD gene aberrations. Further insight into the regulation of CDD activity and its possible relationship to AraC resistance may be achieved by determination of codon 27 allelotypes in significantly larger number of patients and with cloning and analysis of the regulatory regions of the CDD gene.