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Chin Med J (Taipei) 1997;60:184-90.

Expression of DNA Topoisomerase II alpha and Multidrug Resistance p-Glycoprotein in Acute Leukemia

Chang-Fang Chiu1, Kuan-Chih Chow2, Fang-Min Lin1, Chung King Lin1, Shian-Min Liu3, Kuang Y. Chen2

1Section of Hematology, Department of Internal Medicine; 2Cancer Research Group, Cancer Center; 3Department of Pathology, Veterans General Hospital-Taipei and National Yang-Ming University, Taipei, Taiwan, R.O.C.

Abstract

Background. Drug resistance is a major cause of treatment failure in acute leukemia. Overexpression of multidrug resistance gene and decreased activity of topoisomerase IIa are suggested as two important mechanisms for this resistance.

Methods. We used immunohistochemical method to determine the expressions of both topoisomerase II alpha (topo II alpha) and p-glycoprotein (gp-170) in bone marrow biopsy specimens from 68 cases of acute leukemia. Patients were divided into four groups: (1) leukemia cells with high score for topo IIa and negative for gp-170; (2) leukemia cells with high score for topo II alpha and positive for gp-170; (3) leukemia cells with low score for topo II alpha and negative for gp-170; and (4) leukemia cells with low score for topo II alpha and positive for gp-170. The clinical responses were then followed as routine, and the clinical correlation was evaluated by analysis of variance and Pearson Chi-Square test.

Results. The measure of the single parameter (either topo II alpha or gp-170 alone) did not show a significant difference in the overall survival. However, the complete response rate was much higher in the first group patients whose bone marrow reading score was high in topo II alpha and negative for gp-170 expression. Survival duration increased with the increase in the complete response rate.

Conclusions. Combined parameters of topo II alpha and gp-170 are more useful than any individual parameter for the prognosis of acute leukemia.

[Chin Med J (Taipei) 1997;60:184-90.]

Keywords: acute leukemia, DNA topoisomerase IIa, MDR1

Received: October 29, 1996.

Accepted: August 15, 1997.

Address reprint requests to: Kuan-Chih Chow, Ph.D., Cancer Center, Veterans General Hospital-Taipei, No. 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan, R.O.C.

Introduction

Despite some success in the treatment of childhood acute lymphoblastic leukemia, the long-term prognosis of acute leukemia especially in the elderly group is still poor [1]. Drug resistance has been suggested as the major cause for failure of chemotherapy [2]. The mechanisms by which leukemia cells are or become resistant to chemotherapy remain yet to be determined. One mechanism is the overexpression of multidrug resistance gene (MDR1) [3]. MDR1 encodes a membrane glycoprotein (gp-170) that acts as an ATP-dependent efflux pump, transporting a number of structurally unrelated organic compounds [4]. This can lead to resistance to anthracyclines, vinca alkaloids, and podophyllins, drugs that are important in the treatment of acute leukemia. The overexpression is frequently observed in patients with primary resistance at initial diagnosis, or even in complete remission (CR) [5-7]. In a series of 36 patients of acute leukemia, Sato et al. have shown a poor prognosis for those with high levels of MDR1 RNA transcripts [8]. On the contrary, a preliminary report by Ball et al. [9] did not confirm the prognostic value of gp-170 expression as detected by flow cytometry.

The other mechanism is related to topoisomerases that are important therapeutic targets in cancer chemotherapy. In human cells, two types of DNA topoisomerases (type I, monomer, Mr 100,000 and type II, homodimer, Mr 170,000) have been characterized [10,11]. By changing the topological structure of DNA, both enzymes play important roles in DNA metabolism [12,13]. DNA topoisomerase II (topo II) is a proliferation dependent protein that requires ATP to relax DNA molecules [14,15]. Drug scheduling regimens to induce S phase arrest should increase topo II activity. It enhances DNA strand breakage and cellular cytotoxicity of the DNA topoisomerase active drugs [16,18]. Possible mechanisms of drug resistance include decreased level of enzyme, enzyme degradation, altered enzyme function by post-translation modification and mutation resulting in an altered interaction with drug or aberrant DNA recognition. Furthermore, cells that are actively cycling will be more sensitive reflecting the higher levels of topo II found during G2 and mitosis [19,20]. Proliferating cells that contain higher levels of topoisomerase II were also shown to be more sensitive to topoisomerase II specific poisons than quiescent cells. This phenomenon was demonstrated in a number of culture cell systems [15,20-22].

This is the first study applying an indirect immune peroxidase method to study the expression of both DNA topo II alpha [23,24] and MDR1 [5,25,26] in bone marrow biopsies of acute leukemia. We have not only evaluated their levels in leukemia cells but also correlated them with therapeutic response and prognosis. The combined parameters of topo II alpha and gp-170 were found more useful than any single parameter in evaluating the prognosis of patients with acute leukemia.

Materials and Methods

Patients

Bone marrow biopsy specimens of 68 patients who visited VGH between 1986 and 1991 with acute leukemia were sectioned for an analysis of their expressions of topo II alpha and gp-170. The patients included 48 cases with de novo acute non-lymphoblastic leukemia, 11 cases with lymphoblastic leukemia and 9 cases with blastic crisis from chronic myelocytic leukemia. They were similarly treated in the same category. The immunostain for topo II alpha and gp-170 were done for all patients before chemotherapy. Retrospective evaluation of the differences in age, response rate, and survival duration with different expressions of topo II alpha and gp-170 was done before conventional chemotherapy (e.g., araC and adriamycin regimen). The clinical response was then followed as routine.

Monoclonal Antibodies

Monoclonal antibody (IgM) to topoisomerase II alpha (topo II alpha): A single-step protocol to purify drug-sensitive DNA topo II alpha was developed. ATP and teniposide (VM-26) were used to trap the functional enzyme onto kDNA in the crude nuclear extract from CCRF-CEM and K-562 cells. Non-specific DNA binding proteins were washed off kDNA by different concentrations of NaCl in the presence of ATP and VM-26. The drug-sensitive topo II was subsequently released from kDNA with 0.5 M NaCl in the absence of ATP and drug. The eluted enzyme retained the decatenation activity. A single protein band (topo II alpha, p-170) was resolved by gel electrophoresis. Using this purified topo II antigen, 14 mouse monoclonal antibodies to drug-sensitive topo II were raised. Three of them are suitable for immunohistochemical study in bone marrow. Monoclonal antibody (IgG, C219) to gp-170 [27] was purchased from Centocor Diagnostics (Malvern, PA).

Immunohistochemistry

Dewaxed tissue sections were treated with 3% hydrogen peroxide to inactivate. The slides were then incubated with a monoclonal antibody (to topo II alpha or to gp-170), before further incubation with peroxidase-conjugated goat anti-mouse immunoglobulin [28,29]. Following the chromogenic development in diaminobenzidine, brown precipitates were identified as positive stain. Slides were counter-stained with hematoxylin before examination. Each test batch had a positive and negative control to ensure the quality. Unsatisfactory ones were repeated with section and staining. Because of the uneven distribution of expressions of topo II alpha and gp-170, each slide was scored (following the score system shown in Figure 1) in 4 different "blastic" areas of marrow specimen. Each specimen was read by three persons and the results were averaged to get a more objective reading.

According to the score evaluated, patients were then divided into four groups: (1) leukemia cells with high score for topo II alpha and negative for gp-170; (2) leukemia cells with high score for topo II alpha and positive for gp-170; (3) leukemia cells with low score for topo II alpha and negative for gp-170; and (4) leukemia cells with low score for topo II alpha and positive for gp-170. The clinical responses were followed as routine, and the clinical correlation was evaluated by statistical analysis.

Statistical Analysis

Relationship of the expressions of topo II alpha or gp-170 to quantitative parameters (age, percentage of marrow blasts) was studied by analysis of variance. The relationships to qualitative parameters (sex, response rates) were analyzed by Pearson Chi-Square test (or 2-tail Fisher's exact test when expected number of any cells was smaller than or equal to 5 cases). Survival curves were plotted according to the method of Kaplan-Meier (1958). Survival and remission duration of different groups were compared by the logrank test (Mantel-Cox). The respective influence of different parameters on survival duration was calculated according to the Cox proportioned hazards regression method. All calculations were performed using the BMDP-IL statistical software (Los Angeles, CA).

Results

The average age in the study group was 64.5 +/- 10.8 years (42 patients were older than 60, age 14-78, M: F 47:21). No difference in age was observed when patients were divided by the parameter of topo II alpha expression or gp-170 expression (Table 1). When patients were divided by topo II alpha expression into high (score 2 or 3) and low (score 0 or 1) groups, a significant difference between complete response (CR) rate (p = 0.032) was observed. However, no significance in total response rate [CR + partial response (PR), p = 0.447] or overall survival duration (p = 0.126) was detected (Table 1). When patients were divided by the gp-170 expression into positive (score 1, 2, 3) and negative (score 0) groups, no significant difference in either response rate (CR alone or CR + PR, p = 0.185 and 0.061, respectively) or survival (p = 0.646) was noted. Although better CR rate and survival were noted in the negative group (23 vs. 11%, p = 0.061), the difference was marginal.

None of patients with low topo II alpha expression and high gp-170 (n = 23) reached CR. Nine patients reached PR (39%). Median survival was 3.5 months. Four patients with high topo II and high gp-170 reached CR (36%), and two patients reached PR (18%). Among patients with low topo II and low gp-170, only one reached CR (6%), and seven reached PR (41%). No difference in survival was noted in either group (as shown in Figure 2A and Figure 2B). When patients with high topo II score and negative gp-170 were grouped together as one and compared to the rest of patients, response rate (CR, p = 0.004; CR + PR,p = 0.021) was significantly higher in the former group than in the rest of patients. Difference in the median survival was marginal (10 vs. 4 months, p = 0.052) (Table 1) However, benefit of survival duration was significant (Figure 2C). Although age and percentage of marrow blasts are important for the prognosis of acute leukemia, no difference in expression of topo II alpha or MDR1 was observed when we used age 40 to divide patients into two groups (data not shown).

Discussion

For leukemia, several studies using different in vitro chemosensitivity assays have shown a correlation between the clinical response and the in vitro sensitivity [30-33]. It was suggested that the sensitivity to "topo II targeting drugs" could be augmented by exogenous G-CSF through the elevated topo II activity in leukemia cells [21]. However, when Kaufmann et al. [24] examined human AML, no correlation between topo II alpha expression and drug sensitivity was observed. The difference between in vitro assay and clinical study could be in part due to the marked heterogeneity of topo II alpha expression among leukemia cells in marrow. In part, the use of flow cytometry to measure topo II lphaa expression could be short to determine the cell origin of topo II alpha signal. A better way is to identify that cells expressing topo II alpha and MDR1 are truly leukemia cells. Sheu et al. [23] used this method to study topo II alpha expression in relapsed acute leukemia that failed in etoposide treatment. They came to the same conclusion that topo II alpha expression alone was not enough to determine the drug sensitivity. Other factors that are essential for drug resistance should be considered. A preliminary study by Beck et al. [33] indicated that low topo II alpha expression could be accompanied by the high MDRI gene expression in B-cell CLL. MDR gene expression is a dominant factor in drug resistance [34-37].

In fact, the correlation between drug sensitivity and MDR1 gene expression has been studied very extensively at levels of DNA [38], mRNA [39,40] and gp-170 [26,41]. Although the sensitivity of different methods could count for different results [33-43], most of results indicated that MDR1 gene expression is important for drug resistance in tumor cells. A study by Musto et al. [26] also demonstrated that patients with a subset of leukemia cells expressing gp-170 during complete remission might run a higher risk of early and resistant relapse. Our result not only agreed with their observations, but also showed a worse response rate if patient was topo II alpha negative. However, neither their result nor ours reached significant difference because of the relatively small number of legitimate patients involved in these two studies. In our study, because of the characteristics of hospital, the average age of patients was 64.5, much older than the general population. Also, the positive frequency for MDR1 expression was higher in this study. These three reasons could explain in part why we had poorer treatment results.

Topo II alpha and MDR1 gene expressions are different mechanisms involved in drug resistance. Both involve anthracyclines and epipodophyllotoxins that are important for the acute leukemia treatment [3,19]. We examined the expression of both genes in the same bone marrow biopsy specimens. When the two parameters were used to correlate with the prognosis, a significant difference was found. Measurement of topo II alpha or gp-170 alone did not come to that conclusion, although topo II alpha expression had a strong indication for drug sensitivity (Table 1). Neither of them showed significant difference in overall survival. This is a small series of retrospective study but will help to give a direction in future study regarding resistance to acute leukemia.

Acknowledgments

The authors would like to thank Dr. Chin-Yang Li at Mayo Clinic for providing antibody to topoII alpha, Dr. S.P. Wu for his help in pathologic section and Ms. P.C. Lee for her excellent technical assistance in statistical calculation. This work was supported by VGH-84-03 and DOH85-HR-524.

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Copyright: 1997, Chinese Medical Association (Taipei)