Clinical resistance to topoisomerase-targeted drugs

Biochimica et Biophysica Acta 1400 (1998) 275^288

Review

Clinical resistance to topoisomerase-targeted drugs Anne-Marie C. Dingemans, Herbert M. Pinedo, Giuseppe Giaccone * Department of Medical Oncology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Accepted 27 May 1998

Abstract This review describes topoisomerase (topo)-mediated drug resistance and topo expression in human tissues and cancers. In some in vitro studies a relation has been observed between topo I, IIK or IIL expression and sensitivity to topo inhibitors. Drug resistance to topo inhibitors may, however, be multifactorial. Several topo inhibitors are substrates for drug membrane transporters. As most topo inhibitors are cell cycle specific, disturbances in cell cycle regulation can also confer resistance, and downstream events following DNA damage induced by topo inhibitors may be involved in regulating cell death or survival. Several studies in patient specimens have shown a relation between topo IIK expression and the proliferative state of the tumor, higher topo IIK levels being seen in more highly proliferating tumor types. In contrast, topo IIL appears to be expressed in both proliferating and quiescent cells. Furthermore, higher topo I levels were observed in some tumors when compared to their normal counterparts. In some studies a reduced topo IIK level was seen in samples taken after chemotherapy treatment, as compared with specimens prior to treatment. No unequivocal relation was observed, however, between expression or activity of the topo genes and response to chemotherapy; nonetheless only a few studies have properly addressed this question. This review summarizes the results of the clinical studies performed so far, and analyzes the critical issues in performing studies on patient material. 0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. Keywords: DNA topoisomerase ; Topoisomerase inhibitor; Drug resistance; Chemotherapy; Human tumor

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

276

2.

In vitro studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

276

3.

Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Topoisomerases in solid tumors and hematological malignancies . 3.2. Resistance to topo inhibitors in solid tumors . . . . . . . . . . . . . . . 3.3. Resistance to topo inhibitors in hematological malignancies . . . .

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277 277 279 281

4.

Methodological issues in the assessment of drug resistance markers in clinical samples . . .

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Abbreviations: GST, glutathione S-transferase; LRP, lung resistance-related protein; MDR-1, multidrug resistance gene; MRP, multidrug resistance-associated protein; Pgp, P-glycoprotein; topo, DNA topoisomerase * Corresponding author. Fax: +31 (20) 4444355; E-mail: [email protected] 0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 1 4 1 - 9

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1. Introduction Developments in cancer treatment, although de¢nite, have so far not resulted in a large increase in survival, especially in solid tumors [1]. Drug resistance to cancer chemotherapy is one of the causes of failure of treatment in several cancer types. Among the anticancer drugs, the DNA topoisomerase (topo) inhibitors represent an important group of agents. The ¢rst topo inhibitor, camptothecin, was discovered in 1966 [2], although only more recently topoisomerases have been recognized as major targets of a number of antitumor agents [3,4]. Most of the topoisomerase drugs (class I) act through a stabilization of the drug-induced cleavable complex, and formation of irreversible doublestranded DNA breaks which are toxic for the cell. The stabilization of the cleavable complex by topo inhibitors most likely starts a cascade of events, one of which might be the block of replication forks, ultimately leading to cell death [4]. In contrast to class I, class II drugs, such as suramin and merbarone, interfere with the catalytic function of topoisomerases, but do not trap the cleavable complex. The DNA breakage patterns di¡er from drug to drug and speci¢c nucleotide requirements are needed in order for the ternary complex to form [5]. Among the topo II inhibitors, anthracyclines and epipodophyllotoxins are widely used anticancer drugs both for the treatment of hematological malignancies and common solid tumors, such as breast and lung cancers. New camptothecin derivatives (CPT-11 and topotecan) have recently been introduced in the treatment of several forms of solid tumors. The main focus of this review will be studies in patient material where topo-mediated drug resistance has been investigated, in addition sometimes to other drug resistance mechanisms. 2. In vitro studies Several in vitro studies showed a relation between drug sensitivity and expression of the topoisomerase

284

genes, both in cell lines selected for resistance to topoisomerase inhibitors and in unselected cell lines [6,7]. Higher topo IIK levels were found in cell lines derived from sensitive tumor types (e.g. testis cancer, small cell lung cancer) when compared with relatively resistant tumor types (e.g. bladder cancer, non-small cell lung cancer) [8,9]. Direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs has been recently provided by the isolation of genetic suppressor elements encoding antisense RNA [10]. It remains rather unclear whether both topo II isoforms are equally important as targets for topo II drugs. While abundant evidence is available to link the topo IIK isoform to sensitivity of topo II drugs, only a few studies showed reduced expression of topo IIL in cell lines selected for resistance to topo II inhibitors [11]. In a study on six human acute lymphoblastic leukemia (ALL) cell lines a direct correlation was found between expression of topo IIL and cytotoxicity to doxorubicin and etoposide, but not with topo IIK [12]. This indicates that the exact role of the two topo II isoforms in drug sensitivity to topo II inhibitors is still not settled, and suggests that, at least in ALL, topo IIL may be more important than the K isoform in relation to drug resistance. Topo-mediated resistance can be caused by altered or reduced topoisomerase enzyme activity [13,14], and reduced gene expression can be one of the mechanisms leading to reduced topo II activity. Cell lines resistant to topo I inhibitors due to reduced topo I activity can be hypersensitive to topo II inhibitors because of `compensatory' upregulation of the topo II enzyme [15]. A mechanism possibly responsible for reduced gene expression is reduced gene transcription, due to hypermethylation of the gene, which has been described for both topo IIK and topo I [16,17]. Methylation can modulate transcription, but it can also have profound e¡ects on the activity of both topo enzymes in the way that it may alter the distribution of cleavage sites produced by anticancer drugs in chromatin [18]. An altered enzyme activity can also be caused by mutations in the topoisomerase gene; mutations in both the topo I and the topo II genes

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have been found in cell lines selected for resistance to topoisomerase inhibitors [19]. Reduced activity of carboxylesterase may also represent a mechanism by which resistance can be induced in tumor cells to the topo I inhibitor CPT-11, a semisynthetic camptothecin derivative, which requires conversion to its active metabolite SN-38 by carboxylesterase, for antitumor activity. A relation between carboxylesterase activity and in vitro sensitivity to CPT-11 was observed in lung cancer cell lines (Van Ark-Otte, submitted), whereas this relation was not observed in colon cancer cell lines and xenografts [20], suggesting possible di¡erences between tumor types and tissues. Several topo inhibitors are substrates of drug membrane transporters, such as P-glycoprotein (Pgp) [21] and the more recently described multidrug resistance-associated protein (MRP) [22]. Furthermore, multiple drug resistance mechanisms can be present in resistant cells. Most topo inhibitors are cell cycle speci¢c and act on proliferating cells; therefore, alterations in cell proliferation state or cell cycle disturbances can also end up conferring resistance to topo II inhibitors [23]. Finally, drug resistance may also be due to the alteration in downstream events following DNA damage. In three unselected brain tumor cell lines, the relative resistance to etoposide could not be explained by altered etoposide uptake or altered topo II, but Bcl-2 overexpression or mutant p53, both present in these cell lines, could have been implicated in the prevention of etoposide-mediated apoptosis [24]. In immunocompromised mice, tumors expressing the wild type p53 gene regressed after treatment with Q-radiation or adriamycin, whereas p53 de¢cient or mutant tumors were resistant to the same regimen. These results suggest that p53 may be an important determinant of tumor response to therapy [25]. In HeLa cells selected for resistance to teniposide, attenuation of the induction of the proto-oncogene c-jun was seen, which led to the hypothesis that drug resistant cells may fail to activate signaling pathways that are activated in sensitive cells [26].

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3. Clinical studies Studies of the expression of drug resistance markers, including topos, MDR-1, MRP and others depend on the availability of adequate probes and antibodies. These may allow panels of resistance markers to be investigated in the same sample [27]. For immunohistochemical analysis, a large range of monoclonal antibodies against the topo proteins is now available, some of which can also be applied on para¤n embedded archival material [28^30]. Activity of topo II as determined in tumors and normal tissues does not appear to correlate well with protein expression [31,32]. Furthermore, a large heterogeneity in activity of topo I and topo II was observed in di¡erent tumor types [33]. 3.1. Topoisomerases in solid tumors and hematological malignancies Several studies have shown an association between topo IIK and tumor cell proliferation. Interestingly, a newly discovered antibody (Ki-S1), developed as a proliferation marker for staining para¤n embedded sections, appeared to be targeted to topo IIK [34]. In the multivariate analysis of this study, high Ki-S1 expression was an independent prognostic factor of poor outcome in breast cancer patients [29]. Similar results were found in another breast cancer study, where a higher percentage of topo IIK staining cells with a polyclonal antibody was associated with a poorer prognosis [35]. In 20 patients with invasive breast carcinoma topo IIK expression was assessed by immunohistochemistry using formalin ¢xed, para¤n-embedded material. A good correlation was observed with the expression of the proliferation marker MIB-1 (Ki-67), better than with the S-phase fraction or mitotic index, and no correlation was found with ampli¢cation of c-erbB2 [36]. A similar study was conducted in 46 archival cervical squamous lesions: expression increased from normal through increasing grades of dysplasia to invasive cancer, and good correlation was found between topo II and MIB-1 also in this study [37]. Topo IIK expression, investigated in 230 breast cancer cases by immunohistochemistry, showed no expression in non-malignant tissue, whereas topo IIK expression was correlated with proliferation,

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low cellular hormone receptor content, aneuploidy and c-erbB2 overexpression, all features of a more aggressive phenotype [38]. In another study, 56 breast cancers were assessed for mRNA expression of topo IIK and IIL expression by RNase protection assay. The expression of the two genes was not coordinated and topo IIK expression was correlated with the S-phase fraction (proliferation) and was less widely distributed than the topo IIL expression. Relapse-free survival was predicted by topo IIK expression and lymphonodal status, and survival was worse in patients with high S-phase fraction tumors [39]. In several other tumor tissues a strong correlation was observed between the proliferation marker Ki-67 and topo IIK expression [40,41], whereas in normal tissues topo IIK is expressed exclusively in the proliferating areas [42^44]. Furthermore, a high level of topo IIK was observed in biologically aggressive or rapidly proliferating tumors [43]. In an immunohistochemical study of normal gastric mucosa and several types of gastric cancer a strong correla-

tion was seen between expression of topo IIK and Ki67; furthermore there was an increasing expression going from normal gastric mucosa to gastric adenoma, intestinal-type adenocarcinoma and di¡use-type adenocarcinoma, again suggesting a relation with di¡erentiation grade and proliferation [45]. In another study of normal, in£ammatory and neoplastic conditions of the stomach and colon, a good correlation was found between topo IIK and the expression of Ki-67, but no correlation was observed between topo IIK with stage, grade and survival in 38 colon tumors [46]. In contrast to topo IIK, topo IIL is expressed in both proliferating and quiescent cells, although expression in tumor tissues and proliferating cells was somewhat higher compared to the normal counterparts [43,44]. In non-Hodgkin's lymphoma reversetranscriptase polymerase chain reaction (RT-PCR) analysis and immunostaining revealed higher topo IIK mRNA levels in high grade than in low grade lymphomas; moreover a signi¢cant correlation was

Table 1 Clinical studies in solid tumors Ref.

Cancer type

Na

Methods

Genes

Comments

[56] [57]

Bladder cancer Urothelial cancer

12/0 34/10

RNase protection RT-PCR/L2 m

[58]

Nephroblastoma

8/23

mRNA slot-blot

[59]

Lung cancer

48/0

IHC

topo IIK and IIL topo II MRP, MDR-1 GST-Z Pgp/GPX topo II GST-Z topo II, CAT

[54]

Lung cancer

3/8b

RT-PCT/L-actin

MT, TS topo I

[60]

Several typesc

15c

mRNA dot-blot

topo II

Lower expression after epirubicine failure No di¡erence pre/post treatment No di¡erence pre/post treatment Lower post treatment Higher in treated samples Lower in treated samples No di¡erence pre/post treatment No relation with in vitro cytotoxicity to doxorubicin Resistance correlates with high levels No correlation with response to CPT-11 or topotecan Correlation with clinical response to doxorubicin No correlation with clinical response to doxorubicin Lower in treated samples No di¡erence pre/post treatment No di¡erence pre/post treatment No di¡erence pre/post treatment

MDR-1, GST-Z [52]

Ovarian cancer

21/13

[62]

Ovarian cancer

42/12

Decatenation Relaxation IHC Western

topo II topo I Pgp topo IIK

Pre/post, specimen taken before or after chemotherapy respectively; L2 m, L2 -microglobulin; IHC, immunohistochemistry ; CAT, catalase; MT, metallothionein; TS, thymidylate synthase. a Number of samples taken before and after chemotherapy respectively. b From eight patients, three had tissue obtained at diagnosis and all had tissue obtained at autopsy. c Six breast cancers, three hepatocellular carcinomas, ¢ve metastatic breast cancers and one gastric cancer; all specimens taken prior to adriamycin treatment.

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observed between topo IIK and Ki-67 staining index [47,48], whereas topo IIL was present as strong staining in all non-Hodgkin's lymphomas examined, independent of histological subtype [44]. In a number of solid tumors no correlation was found between expression of the two topo II isoforms [40]. In non-small cell lung cancer di¡erent expression of the two topo II genes was observed, and the mRNA expression of the two topo II isoforms in tumors was lower than in cancer cell lines, which are more rapidly proliferating than typical solid tumors [49]. An immunohistochemical study using a speci¢c monoclonal antibody against topo IIK investigated 17 small cell lung cancer and 24 non-small cell lung cancer specimens [50]: the expression was signi¢cantly higher in small cell than in non-small cell lung cancer, and this might partially explain the higher sensitivity of small cell lung cancer. Higher topo I expression levels were observed in colon [51], ovary [52], prostate [53] and lung [49] tumors when compared to their normal counterparts. In contrast, this was not observed in kidney tumors, suggesting that topo I expression may be tumor speci¢c [53]. In 56 lung cancers, topo I mRNA expression was higher in small cell lung cancers than in non-small cell lung cancers [54]. In 179 human tumors representing 18 tumor types, carboxylesterase activity was analyzed: the highest activity levels were observed in lymphoma, small cell lung cancer and mesothelioma. In addition the in vitro sensitivity to the topo I inhibitor CPT-11, as determined by the human tumor colony forming unit assay in 14 samples, was signi¢cantly correlated with the carboxylesterase activity [55]. 3.2. Resistance to topo inhibitors in solid tumors In contrast to the large number of in vitro studies performed, only a few studies investigated the role of drug resistance genes in response to chemotherapy in solid tumors (Table 1). In super¢cial bladder cancer topo IIK and topo IIL gene expression levels were lower in samples taken following epirubicine treatment failure than in samples taken prior to treatment. No relation was observed between topo IIK expression in samples before treatment and the response to epirubicine, but topo IIL levels were signi¢cantly lower in responders [56].

279

In urothelial cancers glutathione S-transferase (GST)-Z levels were lower in samples taken after chemotherapy than in biopsies taken prior to chemotherapy, whereas no di¡erence was seen in MRP, MDR-1 and topo II expression levels as determined by RT-PCR [57]. In nephroblastoma samples Pgp and glutathione peroxidase mRNA expression, analyzed by slot-blot hybridization were higher after treatment with vincristine and actinomycin D, a topo I and II inhibitor, than in untreated specimens. Topo II levels in treated samples were also lower than in untreated samples, as con¢rmed by RTPCR and immunohistochemistry [58]. Unfortunately, in this study it was unclear whether there was a difference between topo IIK and topo IIL expression. Expression levels of GST-Z were not di¡erent between the two groups. In previously untreated squamous cell lung cancer no relation was observed between topo II levels and sensitivity to doxorubicin as assessed by a short term in vitro cytotoxicity assay on dissociated tumor cells. However, signi¢cant correlations were found between metallothionein and thymidylate synthase and sensitivity to doxorubicin, higher levels resulting in a more resistant phenotype [59]. In a study of 15 fresh tumor specimens, of which 11 breast cancer specimens, a signi¢cant correlation (P 6 0.01) was found between topo II expression detected by RNA dot-blot and clinical response to doxorubicin [60]. Gene ampli¢cation is one of the mechanisms by which overexpression may be induced. The gene encoding topo IIK is localized on chromosome 17q in close proximity to the c-erbB2 gene. In 12% of breast cancer samples with c-erbB2 ampli¢cation, topo IIK was co-ampli¢ed, without signs of isolated ampli¢cation of topo IIK [61]. In contrast, only one out of 86 ovarian cancer samples had c-erbB2 ampli¢cation, and no ampli¢cation or co-ampli¢cation of the topo IIK gene was observed [62]. These results indicate that variations in topo IIK expression levels cannot be explained completely by gene ampli¢cation. Topo I and topo II activities were higher in ovarian cancer, compared to benign or borderline tumors [52]. High topo II activity was more frequently seen in untreated samples than in samples derived from patients treated with cis- or carboplatin combined with cyclophosphamide. This di¡erence was not

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seen for topo I activity. Topo IIK, detected by Western blotting, was expressed in 65% of 54 ovarian tumors, at higher levels in stage IV and grade III tumors [62], but no di¡erence in frequency or expression of topo IIK was seen between samples derived from untreated or treated patients. Topo II activity was signi¢cantly correlated with VM-26 induced cleavable complex formation, and topo II decatenation activity was equal but incompletely inhibited by VM-26 in all tumors. The topo II cleavage pattern in plasmid DNA was the same in all tumors. These observations do not point to qualitative di¡erences in topo II in the di¡erent tumors [63]. Topo I protein expression did not correlate with topo I catalytic activity or cleavable complex formation by camptothecin. These results suggest that analysis of cleavable complex formation may be the best determinant

of the role of topo enzymes in response to topo targeted chemotherapy. Scarce information is available on the frequency and relevance of mutations in the topo genes in clinical material. In one out of 13 small cell lung cancer samples analyzed by SSCP (regions B/DNBS, Tyr804 and N3300), two mutations were found in the topo IIK gene. Transversions at codons 486 (G to A) and 494 (A to G) were seen after treatment with cisplatin-etoposide and thoracic radiotherapy, and these mutations were not present in the sample taken prior to treatment. Whether these mutations conferred resistance is still speculative [64]. The mutation at codon 486 was observed before in two leukemia cell lines independently selected for resistance to amsacrine [65], but the mutation at codon 494 was not described previously. In two recent studies, no muta-

Table 2 Clinical studies in hematological malignancies Na

Method

Genes

Comments

25 19/14 36/0 17/4

Northern/IHC RNase Western Functional

topo I+II, MDR-1, GST-Z topo IIK topo IIK, IIL Pgp

28/32

RT-PCR/GAPDH

MDR-1, MRP topo IIK, IIL topo II topo IIK/IIL topo IIK topo IIL MDR-1, MRP topo IIK, IIL MDR-1, MRP, GST, bcl-2, topo IIK topo IIK GST-K,W,Z

No relation with response to CT No relation with in vitro cytotoxicity No relation with response to chemotherapy Weak correlation with response to chemotherapy Both increased in recurrent relapses IIK reduced in relapses, IIL unchanged No relation with treatment status No relation with response to chemotherapy No relation with in vitro sensitivity Higher in resistant cells Both increased in relapses Both unchanged topo IIK, predicted for poor progressionfree survival and overall survival All samples negative No di¡erence between resistant and sensitive cells All samples negative Low expression in all samples High expression in all samples Both unchanged topo IIK low to undetectable, topo IIL unchanged

Ref.

Tumor type

[67] [70] [71]

Myeloma ALL ALL

[75]

ALL

[72] [73] [74]

ALL AML AML

9/4 41/0 20/19/33b

[75]

AML

14/23

[77]

AML

57/0

RT-PCR/L-actin

[80]

CLL

8/10

Western

[81]

CLL

5/6

IHC RT-PCR/GAPDH

[75]

CLL

5/10

RT-PCR/GAPDH

Decatenation Western Activity RT-PCR/GAPDH

Pgp topo IIK topo IIL, MDR-1, MRP MDR-1, MRP topo IIK, IIL

IHC, immunohistochemistry; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphoblastic leukemia. a Number of samples taken before and after chemotherapy respectively. b Thirty-three samples with unknown treatment status.

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tions were observed by SSCP analysis of regions which contain the mutation sites described in camptothecin resistant cell lines (near codons 361 and 366, 533, and 722 and 729) of the topo I gene in 44 previously untreated non-small cell lung cancer samples [66] and in another 56 lung cancer samples, including 13 small cell lung carcinomas [54]. In the latter study no correlation was found between topoisomerase I expression and resistance to the topoisomerase I inhibitors CPT-11 or topotecan [54]. 3.3. Resistance to topo inhibitors in hematological malignancies Most clinical studies on drug resistance have been performed in hematological malignancies, where the malignant cells are more easily accessible (Table 2). In human myeloma cells the mRNA expression of MDR-1, GST-Z, topo I and topo II was determined by Northern blot analysis [67]. Topo I expression was detected in 8/15 cases, and was not signi¢cantly correlated with the patients' response to chemotherapy. In only one of the 16 patients was topo II detectable and at a low level. The patients were initially treated with melphalan and prednisolone and in six cases with a subsequent regimen of vincristine, adriamycin and dexamethasone. MDR-1 mRNA was not detectable in 25 cases, in agreement with absence of immunocytochemical staining for P-glycoprotein. In most cases (16/21) GST-Z, which is involved in detoxi¢cation of melphalan and cyclophosphamide, was expressed at high levels, both at the mRNA and at the protein levels, but no correlation was found with response to chemotherapy. In this study it was not mentioned which patients received the subsequent regimen and at what time point the specimens were taken. More than 95% of patients with childhood ALL achieve complete remission after treatment with combination therapy and 70% of them are cured. The topo II inhibitor daunorubicin is part of the induction chemotherapy. No Pgp could be detected by immunostaining in patients with relapsed ALL, the daunorubicin accumulation was not decreased, and the resistance could not be reversed by verapamil or cyclosporin-A in patient's lymphoblasts [68]. These results indicate that Pgp does not play a major role in drug resistance in childhood ALL; similar results

281

were also obtained with MRP, where no di¡erence was observed between initially diagnosed and relapsed samples [69]. In 19 untreated and 14 relapsed ALL samples large interpatient variation was found for topo IIK mRNA expression, as detected by a sensitive RNase protection assay, but no di¡erence was seen between untreated and relapsed samples. The topo IIK gene expression positively correlated with the percentage of cells in S- and G2 /M-phase but not with in vitro cytotoxicity to daunorubicin and VM-26 in leukemic cells obtained from patients. A concordance was observed between topo IIK mRNA expression and protein expression, as detected by immunocytochemistry [70]. Interestingly, slightly more LRP positive cells were detected in 30 relapsed ALL cells than in 112 initial ALL samples, and LRP expression was signi¢cantly higher in multiple relapse samples than in initial or ¢rst relapse specimens. LRP expression was related to in vitro cytotoxicity to daunorubicin, but not to VP16, and daunorubicin accumulation was lower in LRP positive samples [69]. LRP expression was a factor 2 higher in childhood acute myeloblastic leukemia (AML) than in ALL samples. Also in adult ALL no correlation was seen between topo IIK or topo IIL expression, as assessed by Western blotting, and response to topo II inhibitors containing chemotherapy [71]. Although the short term complete remission rate was high (70%), the long term disease-free survival in this study was poor which might be explained by low levels of topo IIK and small decreases in drug accumulation observed in these samples. In a study of nine acute lymphoblastic leukemia samples obtained from patients, topoisomerase II activity was assessed by a decatenation assay. As expected, leukemic blasts had more topoisomerase II activity than normal blood cells, but no di¡erence in topoisomerase II activity was observed between samples taken at diagnosis and at recurrence [72]. In this series, four patients' samples were available at diagnosis and at relapse. In adult AML a lack of correlation was reported between topo IIK, detected by Western blotting, and the in vitro or in vivo chemosensitivity to daunorubicin, etoposide and m-AMSA in newly diagnosed patients [73]. As all these patients were treated also with Ara-C, this drug may play a dominant role in

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determining the response to the regimen, which might explain the lack in correlation between topo II levels and response in vivo. Levels of topo IIK and topo IIL were strongly inter-correlated and not different between samples taken at diagnosis or at relapse. In this study alterations in drug accumulation did not explain di¡erences in in vitro sensitivity to daunorubicin. Di¡erential analysis of topo II activities for topo IIK and IIL, performed at two di¡erent pH optima, did not show a relation between topo IIK activity and in vitro sensitivity to anthracyclines and epipodophyllotoxins. Interestingly, however, resistant cells had a higher topo IIL activity, suggesting that topo IIL could be the primary target of topoisomerase inhibitors in AML, and the primary drug resistance target in these cells [74]. In a RT-PCR study of topo gene expression and expression of MDR-1 and MRP in acute and chronic leukemias, the expression of topo IIK was reduced in ¢rst and recurrent relapses of ALL, whereas topo IIL did not vary [75]. Furthermore topo IIK expression was correlated with the expression of cyclin A, pointing at the correlation of topo IIK expression and proliferative activity. However, altered expression of MDR-1 and MRP were found in AML, pointing at a multifactorial origin of multidrug resistance in this disease. In another study of 80 AML samples, no correlation was observed between immunocytochemical detection of MRP and the response to chemotherapy or overall survival, whereas patients with high Pgp expression levels had lower complete remission rates [76]. In 57 untreated AML patients the expression of ¢ve drug resistance parameters was assessed (MDR-1, MRP, GSTZ, bcl-2 and topo IIK), in relation to progression-free survival and overall survival. Low topo IIK predicted for poorer outcome, and this result was retained in a multivariate analysis [77]. Median progression-free survival was 9 versus 19 months in low and high topo IIK expressing cells, and median survival was 12 months versus not reached, respectively. Probably, due to the high complete response rate (88%), none of the parameters analyzed predicted response. As shown in in vitro systems, in a small study of nine AML patients, the treatment with topotecan, a

topoisomerase I inhibitor, temporarily increased the expression of topo IIK, suggesting that combined treatment with topoisomerase I and II inhibitors may give additive e¡ects. However, the increased expression of topo IIK was only about threefold, reached its maximum after 3 days of topotecan treatment and was back to baseline levels by day 5 [78]. Chronic lymphocytic leukemia (CLL) has a variable clinical course and although treatment is initially frequently active, acquired resistance is generally observed. Alkylating agents as chlorambucil are usually active in early stages, but doxorubicin-containing regimens are applied in more advanced stages [79]. Response to treatment is an important prognostic factor for overall survival in CLL. Topo IIK was not detectable by Western blot analysis in CLL samples from eight patients with sensitive and ten patients with resistant disease; these samples expressed also very low PCNA levels. Pgp was positive in some samples but not more than 5% of cells stained positively by immunocytochemistry. Although GST-K and GST-Z levels and GST activity were higher in resistant CLL cells than in sensitive cells, this did not reach statistical signi¢cance [80]. RT-PCR analysis, using GAPDH as standard, revealed high MDR-1 and MRP levels both in untreated and treated samples. Topo IIK levels were low, but expression of topo IIL was high [81,75]. Although mutations in the topo IIK gene were found in ¢ve leukemic cell lines selected for resistance to topoisomerase II inhibitors, none of these mutations were present in 15 childhood relapsed ALL samples, analyzed by SSCP and sequencing [82]. These patients were pretreated with etoposide or teniposide. The SSCP analysis was performed for several hot spot regions: the ATP binding sites, motif A, motif B, motif B/DNBS, and the tyrosine 804 binding site. In another study restriction enzyme analysis was performed to analyze for a G-A mutation at position 1493 of the topo IIK gene in samples from 23 AML patients, who failed to achieve a complete response or had recurrent disease, and in the six most highly resistant specimens the putative ATP binding regions B (topo IIK: aa 1246^1539, topo IIL: aa 1641^2264) was sequenced in addition and no mutations were discovered [73].

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4. Methodological issues in the assessment of drug resistance markers in clinical samples From the studies mentioned above, performed in patients samples, no ¢rm conclusions can be drawn. One of the reasons for this is that often only scarce material is available and therefore functional assays, which may provide more information than expression assays, are not possible. Methods such as activity assays and Western blotting require a large number of cells (100 mg protein, þ 2 mg tumor). Furthermore, for correlation studies with response to chemotherapy, tumor samples taken at the ideal time point immediately prior to chemotherapy are not always available. In addition, the assessment of response to chemotherapy is sometimes impossible, like in the case of advanced ovarian cancer, where chemotherapy is given as adjuvant treatment after debulking surgery. Finally, modern chemotherapy usually consists of combinations of several drugs, which makes di¤cult the study of drug resistance to single agents. A number of studies attempted at investigating the e¡ect of single drugs by performing short term in vitro cytotoxicity assays on cells derived from the patient's tumor [59,70].

In order to study topo-mediated drug resistance, several techniques may be employed (Table 3). Expression may be assessed at the RNA or protein level. For studies of RNA expression, Northern blot and RNase protection have been frequently applied. RNase protection assay is approx. 10^20 times more sensitive than Northern blotting, which makes it more appealing for assessment of tumor material, because its level of expression may be several fold lower than in cell lines. For both Northern blotting and RNase protection assay around 10 mg total RNA is necessary to perform only one test, which makes these repeat assays impossible in small biopsy material, such as bronchoscopic biopsies of lung tumors, which yield an average of 20 mg total RNA. RT-PCR is a much more sensitive method, and therefore its major advantage is the low amount of RNA required: speci¢c transcripts can be detected in as little as 10 pg total RNA. In order to quantitate the expression level, the use of a housekeeping gene may be employed, to which the expression of the desired gene is compared, like usually done for Northern blot and RNase protection. The housekeeping genes most frequently used are L-actin, L2 microglobulin and GAPDH. In addition to the exis-

Table 3 Methods to analyze topo-mediated drug resistance Method RNA Northern blot RNase protection Dot-blot In situ hybridization RT-PCR DNA Southern blot PCR Protein Western blot Immunohistochemistry Functional Catalytic activity Decatenation Relaxation Cleavable complex assay SDS/KCl Alkaline elution Filter elution topo isoform activity

283

Amount

Comments

10 Wg 10 Wg 10 Wg 5 Wm slide 10 pg

Bulk method Bulk method, sensitive Bulk method In situ, laborious, di¤cult Sensitive, control genes, bulk method

10 Wg 10 pg

Bulk method Sensitive, bulk method

25^100 Wg 4 Wm slide, 100 cells/slide

Bulk method Easy, in situ, di¤cult to quantitate

109 cells/5 Wl nuclear extract/1 Wg nuclear protein

No distinction between topo IIK and IIL

106 cells/5 Wg protein

Radioactive labeling prior to treatment Labor intensive Simple, unlabeled cells Distinguishes between IIK, IIL

106 cells

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284

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tence of processed pseudogenes for some of these genes (e.g. L-actin) [83^85], a major disadvantage of this quanti¢cation method is that the expression of housekeeping genes is not as constant as believed. An elegant quanti¢cation method is the PCR-aided transcript titration assay in which an internal recombinant RNA is co-ampli¢ed in the same tube [86], which excludes variations present between separately processed tubes. Immunohistochemistry and Western blotting are used for the detection of protein expression. In several studies topo II antibodies were used, which do not distinguish between the two isoforms [59]. Immunohistochemistry requires relatively little material and can identify heterogeneity within the tumor cell populations. However, quanti¢cation of the results is rather di¤cult. RNA in situ hybridization has the same advantages and disadvantages as immunohistochemistry, although it is a far less handy and more laborious method than immunohistochemistry. Functional assays are probably the most reliable way to represent the topo status and the sensitivity of these enzymes to the drug action. A signi¢cant correlation was observed in ovarian cancer samples between VM-26 induced cleavable complex formation and topo II activity, but topo I cleavable complex formation by camptothecin did not correlate with topo I catalytic activity; in addition, no correlation was observed between topo I protein expression assessed by Western blotting and both the cleavable complex formation and the catalytic activity of topo I [63]. The discordance between functional assays and protein or mRNA expression has also been observed in other studies [31,32,87]. In addition to the requirement of a large amount of tissue to perform activity assays, the stability of the enzyme may also be a critical issue. For topo II, the traditional activity assays cannot distinguish between the two isoforms. In a recent study, however, a method has been described in which the topo II isoforms can be selectively determined using their di¡erent pH optima [74]. The ¢lter-binding assay in which topo-DNA complexes can be detected in leukemia cells is a relatively simple method which does not require radioactive labeling of the cells, in contrast to the SDS/ KCl and alkaline elution assays [88]. Its use in solid tumors has, however, not been attempted. In most solid tumors, cancer cells are admixed

with normal stromal cells and surrounded by normal tissue, often without a clear border. A major disadvantage of all bulk techniques, such as those based on total RNA or protein extraction, is that no distinction can be made between signals derived from tumor cells or normal cells; for very sensitive methods, such as RT-PCR based, low levels of expression of the gene of interest in normal cells can have extreme in£uence on the ¢nal results. This problem may be partly overcome by microdissection techniques, by which normal cells are isolated from the tumor cells as precisely as possible. A method which combines functional results with in situ determination is the `comet assay', which can measure DNA breaks in single cells. The sensitivity of this assay is comparable to that of DNA precipitation and alkaline unwinding assays for detecting DNA strand breaks induced by VP-16 [89]. It has been shown that with this assay drug sensitive cells can be distinguished from drug resistant cells [90]. The applicability of this technique to patient material and solid tumors in particular, needs to be worked out.

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