Clinical Cancer Research Targets Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 14, 1759, March 15, 2008. doi: 10.1158/1078-0432.CCR-07-1395
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doussis-Anagnostopoulou, I. A.
Right arrow Articles by Pangalis, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doussis-Anagnostopoulou, I. A.
Right arrow Articles by Pangalis, G. A.

Imaging, Diagnosis, Prognosis

Topoisomerase II{alpha} Expression as an Independent Prognostic Factor in Hodgkin's Lymphoma

Ipatia A. Doussis-Anagnostopoulou1, Theodoros P. Vassilakopoulos2, Irini Thymara3, Penelope Korkolopoulou3, Maria K. Angelopoulou2, Marina P. Siakantaris2, Styliani I. Kokoris2, Evangelia M. Dimitriadou2, Christina Kalpadakis2, Marina Matzouranis2, Loukas Kaklamanis1, Panayiotis Panayiotidis2, Marie-Christine Kyrtsonis2, Athina Androulaki3, Efstratios Patsouris3, Christos Kittas1 and Gerassimos A. Pangalis2

Authors' Affiliations: 1 Laboratory of Histology and Embryology, 2 First Department of Internal Medicine and Department of Haematology and 3 Department of Pathology, National and Kapodistrian University of Athens, Medical School, Athens, Greece

Requests for reprints: Ipatia Doussis-Anagnostopoulou, Laboratory of Histology and Embryology, Athens Medical School, 75, Micras Asias str., 11527 Goudi, Athens, Greece. Phone: 0030-210-7462348/0030-210-6717049; E-mail: ipatiada{at}med.uoa.gr.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: To correlate the immunohistochemical expression of topoisomerase II{alpha} (topoII{alpha}) in Hodgkin's lymphoma (HL) with clinicopathological parameters, the expression of Ki-67 and the outcome of patients, who had been homogenously treated with ABVD or equivalent regimens.

Experimental Design: Immunohistochemistry using the monoclonal antibody Ki-S1 (topoII{alpha}) was performed in 238 HL patients. MiB1 (Ki-67) expression was evaluated in 211/238.

Results: The mean ± SD percentage of topoII{alpha}- and Ki-67–positive Hodgkin-Reed-Sternberg (HRS) cells was 63 ± 19% (5%-98%) and 73 ± 19% (8%-99%), respectively. The median percentage of topoII{alpha}-positive HRS cells was 64% (interquartile range, 51-78%). There was no correlation between topoII{alpha} expression and patient characteristics. TopoII{alpha} and Ki-67 expression were correlated (Spearman's Rho 0.255, P < 0.001). TopoIl{alpha} expression within the highest quartile of this patient population was predictive of failure free survival (FFS) (10-year rates 82 ± 3% vs 68 ± 7%, P = 0.02 for patients falling into the quartiles 1-3 and 4 respectively). In multivariate analysis topoII{alpha} expression was independently predictive of FFS.

Conclusion: TopoII{alpha} was expressed in all cases of HL showing a correlation with Ki-67 expression. Under current standard therapy including drugs inhibiting its activity, topoII{alpha} was an independent adverse predictor of FFS with no statistically significant correlation with other established prognostic factors.

Topoisomerases are enzymes that relax DNA supercoiling arising in a variety of nuclear processes. The mechanism of action of topoisomerases I and II involves the formation of transient single or double-stranded DNA breaks respectively. Topoisomerase II is found in two isoforms, namely topoisomerase II{alpha} (topoII{alpha}) and topoisomerase IIβ. TopoII{alpha} is a target for several cytotoxic agents used in the treatment of hematologic malignancies, such as doxorubicin, epirubicin, mitoxantrone, etoposide, and teniposide (1–3). These agents appear to stabilize the DNA-topoII{alpha} complex and inhibit DNA relegation, resulting in an accumulation of lethal double-strand DNA breaks (4). Experimental studies have shown that cells expressing high levels of topoII{alpha} are drug sensitive, while those with low levels are drug resistant (5), making the enzyme a potentially useful predictor of tumour sensitivity to chemotherapy.

Interestingly, topoII{alpha} is mainly expressed in proliferating cells (2), being identical to the proliferation-associated antigen KiS1 (6), and can be used as a proliferation marker in normal and neoplastic cells (7). TopoII{alpha} expression demonstrates a positive correlation with the widely used proliferative marker Ki67 in a variety of normal and neoplastic tissues (8–11).

The dual role of topoII{alpha} makes it an interesting subject of investigation in order to explore the association of its expression with the outcome of patients with neoplastic disorders. Thus it is not yet clear whether high levels of topoII{alpha} are beneficial, increasing tumor sensitivity to topoII{alpha} inhibitors, or they are associated with worse prognosis reflecting increased proliferative activity. Clinical results in the literature are so far contradictory. While in some studies high levels of topoII{alpha} have been associated with chemosensitivity, especially in anthracycline treated breast cancer (12–14), in others topoII{alpha} expression was associated with inferior outcome (15–19). In various subtypes of non-Hodgkin's lymphomas, high topoII{alpha}/Ki67 ratio and high topoII{alpha} expression independently predicted for inferior outcome (20–22).

In a recent report on 42 cases of Hodgkin's lymphoma (HL), low levels of topoII{alpha} expression were associated with shorter survival (23), but this was not confirmed by a preliminary study of 57 patients from our group (24). Both these studies were small and used different patient selection criteria as well as different cuttofs for topoII{alpha} dichotomization. In view of these conflicting data, we extended our observations on topoII{alpha} expression in a much larger series of 238 patients with HL, who had been homogenously treated with topoII{alpha} inhibitors, including chemotherapy based on ABVD or equivalent regimens with or without radiotherapy. TopoII{alpha} expression was correlated with clinicopathological parameters, the expression of Ki67 and patient outcome.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patients and staging. We analyzed 238 patients with HL, who were diagnosed and treated at the Haematology Section, First Department of Internal Medicine, "Laikon" General Hospital, National and Kapodistrian University of Athens. All patients were older than 14 years, were HIV-negative, had pretreatment archival lymph node tissue material available for topoisomerase II{alpha} immunostaining and had received treatment with anthracycline-based chemotherapy with or without radiotherapy. Approval was obtained by the appropriate Institutional Review Board. All histologic material was reviewed and classified according to the recent WHO classification (25). The baseline patient characteristics, median follow-up, and failure free survival rates were comparable with those of patients who had also received anthracycline-based chemotherapy with or without radiotherapy during the same period, but had not tissue material available for topoisomerase II{alpha} immunostaining (all P-values >0.05; data not shown).

All patients were clinically staged according to the Ann-Arbor system (26), using standard staging procedures. Clinical stages IA and IIÁ were considered early, while clinical stages IB, IIB, III and IV were considered advanced. Anemia was defined as the presence of hemoglobin levels <13g/dl for males and <11.5g/dl for females (27). Serum albumin and severe lymphocytopenia were analyzed at the International Prognostic Score (IPS) cut-offs of <4g/dl and <0.6 x 109/l or <8% respectively (28). The number of involved anatomic sites was determined as previously described (29).

Treatment strategies. Treatment strategies for early Ann-Arbor stage (AAS IA, IIA) and advanced stage (IB, IIB, III, IV) patients have been described previously (30). Early stage patients were scheduled for combined modality therapy including low-dose involved field radiotherapy. Advanced stage patients received chemotherapy. Radiotherapy was administered to 68% of patients with AAS IB, IIB and IIIA. In contrast only 26% of patients with AAS IIIB and IV received radiotherapy. ABVD or EBVD was administered to 82% of the patients, alternating MOPP/ABVD or MOPP/EBVD to 12% and MOPP/ABV or MOPP/EBV hybrid to 6%. All these regimens are currently considered equivalent (31–35).

Immunohistochemical staining. Immunohisthochemical staining was performed as previously described (36). The monoclonal antibodies Ki-S1 (DAKO, Denmark; dilution 1:50), which recognises the topoII{alpha} isophorm and MIB1 (YLEM; dilution 1:100) for Ki67 were used. Omission of the primary antibody was used as a negative control in all cases, while a reactive lymph node was used as a positive control.

Nuclei from at least 100 neoplastic Hodgkin Reed-Sternberg (HRS) cells were to be evaluated in each case. This was possible in approximately 90% of the cases, while in the remaining cases we evaluated as many HRS cells as possible (usually >80). The labelling index (LI) was calculated as the percentage of positive cells. All nuclear staining, whether weak or strong, was counted as positive. The evaluation of topoII{alpha} expression was blinded, since it was performed without knowledge of patients' clinical data and outcome. Evaluation of MIB1 (Ki67) expression was also performed without knowledge of the topoII{alpha} status and patients' clinical data and outcome.

The intraobserver and interobserver variability in the evaluation of topoII{alpha} expression was assessed in a subgroup of 28 patients by IADA and PK.

Statistical analysis. The mean percentages of topoII{alpha} expressing HRS cells among various subgroups of patients were compared by the Student's t-test or the one-way analysis of variance (one-way ANOVA). The intraobserver and interobserver variability of topoII{alpha} expression was evaluated by the Pearson's correlation coefficient. The correlation between topoII{alpha} and Ki67 expression was evaluated by Spearman's rho coefficient, given that the distribution of Ki67 deviated from normality. Failure-free survival (FFS) was defined as the time interval between treatment initiation and treatment failure or last follow-up. Failure was defined as inability to achieve complete or partial remission (CR, PR) during initial therapy, requiring switch to another chemotherapy regimen, death during initial therapy, or progression after an initial CR or PR. Overall survival (OS) was defined as the time interval between treatment initiation and death of any cause or last follow-up. Survival curves were plotted by the method of Kaplan-Meier. The identification of prognostic factors in univariate analysis was based on the log-rank test. Multivariate analysis was performed using Cox's proportional hazards model. A backward stepwise selection procedure, with entry and removal criteria of P = 0.05 and P = 0.10, respectively, was used. In order to avoid the use of arbitrary cutoffs, we performed survival analysis according to the quartiles of topoII{alpha} expression. High topoII{alpha} expression was defined as percentages of topoII{alpha}-positive HRS cells falling into the upper quartile (designated as Q4) vs. those falling within the three lower ones (designated as Q1-3). TopoII{alpha} expression was also evaluated as a continuous covariate (percentage of topoII{alpha}-positive HRS cells in each case) in multivariate analysis. Furthermore, topoII{alpha} expression was evaluated according to the cutoff obtained by a ROC curve-based approach. Finally, topoII{alpha} expression was tested against the IPS value in multivariate analysis (28); all individual IPS variables were excluded from this analysis.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
Patients' characteristics
Patients' clinical and laboratory characteristics are summarized in Table 1 . They were compatible with other reported unselected series of patients with non-pediatric HL. The median age of the patients was 30 years (15-82) and 117 (49%) were males. Histologically, there were 232 cases of classical HL (97%) and 6 cases of nodular lymphocyte predominance (NLP) HL (3%). In detail, 174 patients were classified as nodular sclerosis (NS) subtype (73%), 47 cases as mixed cellularity (MC) (20%), 8 as lymphocyte rich (3%), 1 as lymphocyte depletion (<1%), 1 as interfollicular (<1%) and 1 as classical HL, unclassified (<1%). The median follow-up of patients, who were alive at the time of the analysis, was 111 months (23-222).


View this table:
[in this window]
[in a new window]

  		Table 1. Correlation of the percentage of topoisomerase II{alpha}-positive HRS cells with baseline characteristics of patients with Hodgkin's lymphoma

 
Evaluation of topoII{alpha} and Ki67 expression
Immunohistochemical expression of topoII{alpha} was seen in all cases and was mainly present in the neoplastic HRS cells and variants. A number of reactive small lymphocytes and larger cells within residual germinal centers were also positive. The staining observed was diffuse nuclear (Fig. 1A ), with an occasional punctate pattern. The percentage of positive HRS cells ranged from 5% to 98%, following an approximately normal distribution with a mean ± SD of 63 ± 19% and a median value of 64% (interquartile range, 51-78%). Both intraobserver and interobserver variability were very low with Pearson's correlation coefficients of 0.943 (P < 0.001) and 0.950 (P < 0.001) respectively.


Figure 1
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. A, topoisomerase II{alpha} expression was seen in all cases and was mainly present in the neoplastic HRS cells and variants, as a diffuse nuclear staining. B, C, D, effect of topoisomerase II{alpha} expression on the outcome of patients with Hodgkin's lymphoma treated with ABVD or equivalent regimens with or without radiotherapy: (B) Failure Free Survival (FFS) in all patients; (C) FFS in early clinical stages (IA and IIA); (D) FFS in advanced clinical stages (IB, IIB, III and IV).

 
No statistically significant relationship was found between topoII{alpha} expression and demographic, clinical, and conventional laboratory parameters including IPS, with the exception of gender, where males had marginally higher expression than women (P = 0.08; Table 1). Although the 6 patients with NLP had somewhat higher mean percentage of topoII{alpha} expression than patients with classical HL (75 ± 17% vs. 63 ± 19%), the difference was not statistically significant (P = 0.16). TopoII{alpha} expression did not also differ according to the subtypes of classical HL (Table 1).

Ki67 expression was evaluated in 211/238 patients (89%). The staining was diffuse nuclear and the mean ± SD percentage of positive HRS cells was 73 ± 19% (range 8%-99%). The median Ki67 expression was 76% (interquartile range, 61-88%). A rather loose positive correlation was found between TopoII{alpha} and Ki67 expression (Spearman's rho 0.255, P < 0.001).

Univariate analysis
Remission rates. Remission rates did not differ according to topoII{alpha} expression: Primary refractory disease was observed in 4.5% vs. 3.5% of patients with topoII{alpha} expression within Q1-3 and Q4 respectively (P = 0.73). Furthermore, early failure, defined as progression or relapse within the 1st year from diagnosis, was observed in 7.8% vs. 5.1% of patients respectively (P = 0.48).

Failure-free survival in all patients. The results of univariate analysis of FFS are summarized in Table 2 . Most of the established conventional prognostic factors proved to be statistically significant. Except of quartile analysis, a ROC curve-based approach was used to dichotomize topoII{alpha} values. According to the latter a cutoff of 74.8% was suggested, which was rounded to 75%.


View this table:
[in this window]
[in a new window]

  		Table 2. Univariate analysis of prognostic factors in 238 patients with Hodgkin's lymphoma

 
The 10-year FFS was 82 ± 3% vs 68 ± 7% for patients with topoII{alpha} expression within Q1-3 and Q4, respectively (P = 0.02) (Fig. 1B and Table 2). Using 75% as ROC-based cutoff for topoII{alpha} expression, the discrimination was even better with 10-year FFS being 84 ± 3% vs 67 ± 6% (P = 0.002). In this analysis 72 patients (30%) fell into the adverse group.

Failure-free survival in stage and IPS subgroups. Among 127 patients with early stage HL (IA/IIA), topoII{alpha} expression did not show a statistically significant association with FFS (10-year rates 89 ± 3% vs 83 ± 7%, P = 0.36 for patients with topoII{alpha} expression within Q1-3 and Q4 respectively; Fig. 1C). In contrast, topoII{alpha} expression was significantly associated with FFS among the 111 patients with advanced disease (10-year rates 74 ± 5% vs 52 ± 11%, P = 0.03 for patients with expression within Q1-3 and Q4; Fig. 1D). When patients were stratified according to the IPS, topoII{alpha} expression had again a non-significant effect on FFS in patients with IPS <3 [10-year rates 82 ± 4% vs 76 ± 8%, P = 0.64 for patients with expression within Q1-3 and Q4 respectively], and a highly significant effect on FFS of patients with IPS ≥3 [corresponding 10-year rates 74 ± 8% vs 30 ± 14%, P = 0.0008], so that patients with IPS ≥3 and high topoII{alpha} expression had a particularly poor outcome.

Failure-free survival in treatment modality subgroups. High topoII{alpha} expression was highly predictive of adverse outcome in patients treated with chemotherapy only; 10-year FFS rates were 71 ± 7% vs. 29 ± 15% in patients falling within Q1-3 vs. Q4 respectively (P = 0.003). Among 69 patients who received chemotherapy alone, 56 (81%) had advanced stage disease. On the contrary, the effect of high topoII{alpha} expression in patients treated with combined modality was much smaller: 10-year FFS rates were 88 ± 3% vs. 82 ± 6% in patients falling within Q1-3 vs. Q4 respectively (P = 0.25). Among 169 patients treated with combined modality, 114 (67%) had early stage disease.

Overall survival. During follow-up 33 deaths were recorded: 20 (61%) due to HL-related and 13 (39%) due to unrelated causes.

The difference in overall survival was borderline: Patients with topoII{alpha} expression within Q1-3 had a 88 ± 3% 10-year overall survival rate vs. 81 ± 5% for those with expression within Q4 (P = 0.06). At the 75% ROC-based cutoff the overall survival difference was significant (10-year rates 90 ± 3% vs. 79 ± 5%, P = 0.02).Patients with topoII{alpha} expression within Q1-3 had a 94 ± 2% 10-year cause-specific survival rate vs 83 ± 5% for those with topoII{alpha} expression within Q4 (P = 0.005). The discriminative ability at the 75% cutoff was even better (10-year rates 96 ± 2% vs. 80 ± 5%, P = 0.0003).

Multivariate analysis
TopoII{alpha} expression was evaluated together with covariates, which were significant in univariate analysis and had <10% missing values, i.e. stage IV, B-symptoms, and number of involved anatomic sites. The results of multivariate FFS analysis are summarized in Table 3 . High topoII{alpha} expression (within Q4) had independent prognostic significance for FFS, along with B-symptoms and, marginally, the number of involved anatomic sites. When covariates with >10% missing values were included, topoII{alpha} expression within Q4, stage IV, and reduced serum albumin levels remained independent prognostic factors for FFS.


View this table:
[in this window]
[in a new window]

  		Table 3. Multivariate analysis of prognostic factors for failure-free survival in 238 patients with Hodgkin's lymphoma

 
When the percentage of topoII{alpha}-positive HRS cells was evaluated as a continuous covariate, its independent prognostic significance for FFS persisted: Increasing topoII{alpha} expression (P = 0.02; relative risk 1.020 per percentage unit; 95% CI 1.004-1.036), B-symptoms and the number of involved anatomic sites were again selected in the multivariate model.

High topoII{alpha} expression (within Q4) remained an independent prognostic factor for FFS after adjustment for the IPS (P = 0.02; Table 3). The same was true when topoII{alpha} expression was evaluated as a continuous covariate (P = 0.03; relative risk 1.019 per percentage unit; 95% CI 1.002-1.036). When B-symptoms and the number of involved anatomic sites were evaluated along with topoII{alpha} and IPS, the significance of topoII{alpha} persisted, while that of IPS and B-symptoms were borderline.

When topoII{alpha} expression was analyzed at the ROC-based cutoff of 75%, its significance was strengthened in all multivariate models.

In multivariate analysis of overall survival topoII{alpha} had a non-significant effect after adjustment for age and the number of involved sites (P = 0.13).

Prognostic significance of Ki67 and TopoII{alpha}/Ki67 ratio
In the population of 211 patients with available data on Ki67 expression (with 43 failure events), high topoII{alpha} expression (within Q4) was again associated with inferior FFS (P = 0.03). Irrespectively of the cutoff value used, Ki67 expression was not associated with FFS. In contrast, the ratio topoII{alpha}/Ki67 was of prognostic significance: The 10-year FFS was 82 ± 3% for the 147 (70%) patients with ratios <1 vs. 72 ± 6% for the 64 (30%) patients with ratios ≥1 (P = 0.03). In multivariate analysis, a topoII{alpha}/Ki67 ratio ≥1 was independently associated with FFS (P = 0.04) along with B-symptoms (P = 0.03) and, marginally, stage IV (P = 0.06). However, neither Ki67 nor the ratio topoII{alpha}/Ki67 added independent prognostic information in multivariate models already including the percentage of topoII{alpha} expression.


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
The expression of topoII{alpha} in human neoplasia has been the subject of many recent reports. In this study all cases of HL expressed topoII{alpha} with a median number of positive HRS cells of 64%. These results are in accordance with those previously described by us in a different patient series (37). Brown et al., in series of 49 cases of HL, found similar percentages of topoII{alpha}-positive neoplastic cells in cases of classical HL (58% in NS and 68% in MC), while the percentage of topoII{alpha}-positive neoplastic cells in 15 cases of LPHL was 84% (38). This result was obtained from the evaluation of CD20/topoII{alpha} double positive cells. Although the mean percentage of topoII{alpha}-positive neoplastic cells in our NLP HL patients was numerically higher than the one observed in classical HL, the difference did not reach statistical significance. However the number of cases of NLP HL was low to permit a reliable statistical analysis. In contrast to the high topoII{alpha} expression observed in the present, as well as other series (37, 38), other investigators have reported lower median percentages of positive HRS cells in the order of 30% (23, 39). These differences may be related to the use of different monoclonal antibodies for topoII{alpha} detection, as it has been demonstrated that KiS1 is an antibody with high binding affinity to topoII{alpha} (40).

We demonstrated here that high topoII{alpha} expression had independent adverse prognostic significance in a series of 238 patients with HL, who had been treated with ABVD or equivalent regimens with or without RT in a single center. This treatment approach invariably included the administration of doxorubicine or epirubicine, which exert their antineoplastic action through topoII{alpha} inhibition. Our results contradict those reported by Provencio et al. (23), who had suggested a favorable prognostic impact of higher topoII{alpha} expression in a series of 42 patients with advanced HL. However, a more recent report by the Spanish group demonstrated that high topoII{alpha} expression (within the corresponding Q4) was associated with inferior cause-specific survival in a series of 235 patients, eventhough the difference did not reach statistical significance (41). The absolute difference in long-term cause-specific survival between patients with topoII{alpha} expression within Q1-3 and Q4 in the latter study was approximately 8-10% (approximation obtained from survival curves), a figure consistent with our data. Our results are also in agreement with the adverse significance of topoII{alpha} expression in other neoplasms (15, 16, 20–22, 42–44), including non-Hodgkin's lymphomas (20–22), even after chemotherapy with topoII{alpha} inhibitors (18–20).

A simplified explanation of our findings could be that topoII{alpha} exerted its effect on the prognosis of HL merely through its role as a proliferation marker, which is further supported by the positive correlation that we found both between topoII{alpha} and MIB1 (Ki-67) expression. The mean percentage of Ki67-positive cells was higher than that of TopoII{alpha} positive cells, a finding described both in our previous study (37) and in non-Hodgkin's lymphomas (20, 45). However topoII{alpha} might provide a better estimate of the number of cycling cells than Ki67, because it is selectively present during the later phases of the cell cycle (40, 46, 47). Thus the data presented here are compatible with a role of topoII{alpha} as a proliferation marker.

In contrast to topoII{alpha}, Ki-67 expression was of no prognostic significance in our series. Although Ki67 expression was predictive of the outcome in the initial study of the Spanish group (48), this was not confirmed in a subsequent larger series evaluated by tissue arrays (49). Additionally, in accordance with a previously report of our group on non-Hodgkin's lymphomas (20), a ratio of topoII{alpha}/Ki67 ≥1 was an independent predictor of inferior FFS in this series as well, but did not add prognostic information independent of topoII{alpha} expression per se.

Except of a borderline association with male gender, topoII{alpha} expression was neither correlated with any of the examined conventional demographic, clinical, histologic, and laboratory parameters nor with the value of IPS. Thus topoII{alpha} does not appear to be a surrogate marker of either tumor burden or the biological aggressiveness of the disease and may actually represent a ‘‘primary’' prognostic variable. This is in contrast with most conventional prognostic factors, which present extensive interrelationships (28), and resembles to what has been observed with a small number of recently described biological markers, such as bcl-2 (34) and activated caspase-3 expression (50).

Given the lack of correlation with other prognostic factors and its prognostic significance in univariate analysis of FFS, topoII{alpha} emerged as an independent prognostic marker in multivariate analysis. Using the upper quartile as a definition for high topoII{alpha} expression we avoided '‘data fitting'', which could arise by the introduction of arbitrary or ‘‘best’' cutoffs. The validity of our findings is strengthened by the fact that topoII{alpha} was not only predictive of FFS at the cut-off of the upper quartile, but also carried independent prognostic significance when evaluated as a continuous covariate. A ROC-curve defined cutoff (75%) led to the demonstration of an even stronger prognostic impact of high topoII{alpha} expression.

Despite its clear independent prognostic significance, we further tested whether topoII{alpha} added to the prediction achieved by the IPS, which is the most popular prognostic index for advanced HL (28), while very similar systems may also work in localized disease (51–53). Thus in another multivariate approach including both IPS and topoII{alpha}, the latter proved again to be an independent prognostic factor for FFS.

The adverse prognostic effect of high topoII{alpha} expression was mainly restricted to advanced stage patients, while its effect in early stages was not statistically significant. Furthermore, high topoII{alpha} expression was predictive of adverse outcome in patients treated with chemotherapy only, a finding suggesting that high topoII{alpha} expression did not confer sensitivity to anthracycline-based chemotherapy. However, since 81% of patients treated with chemotherapy alone had advanced stage disease, it is not clear that the highly significant difference was related to the applied treatment or to the inclusion of advanced stage patients. On the contrary, the effect of high topoII{alpha} expression in patients treated with combined modality was much smaller, probably due to the fact that most patients treated with combined modality had early stage disease, resulting to a low number of failure events, which reduced the power of the analysis to demonstrate any prognostic effect of topoII{alpha}.

TopoII{alpha} is necessary as a substrate for anthracyclines and other drugs, in order to exert their antineoplastic activity. A simplified approach could suggest that if more topoII{alpha} is expressed in the HRS cells, the target for anthracyclines would be more abundant and the antitumor activity would be greater. This view has been supported by recent results, especially in breast cancer, but in others tumors as well (12–14, 23). However, as more HRS cells express topoII{alpha}, the amount of anthracyclines delivered by standard chemotherapy regimens may not suffice to substantially inhibit topoII{alpha} activity and lead the neoplastic cells to apoptosis. This hypothesis offers an additional explanation of our findings. Furthermore, if this is indeed the major mechanism responsible for the higher failure rates found in patients with higher topoII{alpha} expression, then it might have therapeutic implications: it would be logical to hypothesize that patients with high levels of topoII{alpha} might benefit from dose intensification of topoII{alpha} inhibitors or from the addition of a second topoII{alpha} inhibitor in the initial chemotherapy regimen. This is the case with BEACOPP-escalated, the most effective but also toxic regimen available for advanced HL (54), which is now also being tested in patients with early stages and unfavorable prognostic profile (55). BEACOPP-escalated maintains the dose intensity of doxorubicine in comparison with ABVD, but—in addition—includes 3 days of etoposide, another topoII{alpha} inhibitor, at high dose of 200 mg/m2 daily. It would be very interesting to see whether the prognostic significance of topoII{alpha} expression will be overcome by the administration of BEACOPP-escalated, a situation that would strengthen our hypothesis.

In conclusion, we demonstrated the adverse prognostic significance of topoII{alpha} expression in patients with HL treated with ABVD or equivalent regimens with or without radiotherapy. Apart from circulating factors including cytokines (27, 56–58), molecules involved in apoptosis (34, 49, 56) and polymorphisms implicated in drug metabolism (59), topoII{alpha} expression appears to have also a place in the armamentarium of biological prognostic factors in HL due to its implication in cell proliferation and drug sensitivity mechanisms. Following appropriate validation, this molecule may offer a biological rational for treatment intensification with topoII{alpha} inhibitors, generating an hypothesis that could be tested in future trials.


   Footnotes
 
Grant support: IASIS, a nonprofit organization raising funds for research in leukemias, lymphomas, and related disorders.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: I.A. Doussis-Anagnostopoulou and T.P. Vassilakopoulos contributed equally to this work and both should be considered as first authors.

Received 6/ 7/07; revised 10/ 2/07; accepted 10/17/07.


   References
Top
 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

  1. Schneider E, Yaw-Huei H, Liu LF. DNA topoisomerases as anticancer drug targets. Adv Pharmacol 1990;21:149–83.[Medline]
  2. Drake FH, Hofmann GA, Bartus HF, Mattern MR, Crooke ST, Mirabelli CK. Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. Biochemistry 1989;28:8154–60.[CrossRef][Medline]
  3. Robinson MJ, Osheroff M. Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II. Biochemistry 1991;30:1807–13.[CrossRef][Medline]
  4. Zhang H, D'Arpa PD, Liu LF. A model for tumor cell killing by topoisomerase poisons. Cancer Cells 1990;2:23–7.[Medline]
  5. Lage H, Helmbach H, Dietel M, Schadendorf D. Modulation of DNA Topoisomerase II{alpha} activity and expression in melanoma cells with acquired drug resistance. Br J Cancer 2000;82:488–91.[CrossRef][Medline]
  6. Boege F, Andersen A, Jensen S, Zeidler R, Kreipe H. Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase-II{alpha}. Am J Pathol 1995;146:1302–8.[Abstract]
  7. Turley H, Comley M, Houlbrook S, et al. The distribution and expression of the two isophorms of DNA topoisomerase II in normal and neoplastic human tissues. Br J Cancer 1997;75:1340–6.[Medline]
  8. Lynch BJ, Guinee DG, Holden JA. Human DNA topoisomerase II-alpha: a new marker of cell proliferation in invasive breast cancer. Hum Pathol 1997;28:1180–8.[CrossRef][Medline]
  9. Ito K, Sasano H, Yabuki N, et al. Immunohistochemical study of Ki-67 and DNA topoisomerase II in human endometrium. Mod Pathol 1997;10:289–94.[Medline]
  10. Ohashi Y, Sasano H, Yamaki H, et al. Topoisomerase IIalpha expression in esophageal squamous cell carcinoma. Anticancer Res 1999;19:1873–80.[Medline]
  11. Costa MJ, Hanses CL, Holden JA, Guinee D, Jr. Topoisomerase IIalpha: prognostic predictor and cell cycle marker in surface epithelial neoplasms of the ovary and peritoneum. Int J Gynecol Pathol 2000;19:248–57.[CrossRef][Medline]
  12. MacGrogan G, Rudolph P, Mascarel Id I, et al. DNA topoisomerase IIalpha expression and the response to primary chemotherapy in breast cancer. Br J Cancer 2003;89:666–71.[CrossRef][Medline]
  13. Durbecq V, Paesmans M, Cardoso F, et al. Topoisomerase-II{alpha} expression as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel. Mol Cancer Ther 2004;3:1207–14.[Abstract/Free Full Text]
  14. Koshiyama M, Fujii H, Kinezaki M, Yoshida M. Correlation between TopoIIalpha expression and chemosensitivity testing for TopoII-targeting drugs in gynaecological carcinomas. Anticancer Res 2001;21:905–10.[Medline]
  15. Holden JA, Townsend JJ. DNA topoisomerase II-alpha as a proliferation marker in astrocytic neoplasms of the central nervous system: correlation with MIB1 expression and patient survival. Mod Pathol 1999;12:1094–100.[Medline]
  16. Rudolph P, MacGrogan G, Bonichon F, et al. Prognostic significance of Ki-67 and topoisomerase II{alpha} expression in infiltrating ductal carcinoma of the breast. A multivariate analysis of 863 cases. Breast Cancer Res Treat 1999;55:61–71.[CrossRef][Medline]
  17. Grandgirard N, Ly-Sunnaram B, Ferrant D, et al. Impact of topoisomerase II{alpha}lpha and spermine on the clinical outcome of children with acute lymphoblastic leukemia. Leuk Res 2004;28:479–86.[CrossRef][Medline]
  18. Dingemans AMC, van Ark-Otte J, Span S, et al. Topoisomerase II{alpha} and other drug resistance markers in advanced non-small cell lung cancer. Lung Cancer 2001;32:117–28.[CrossRef][Medline]
  19. Dingemans AM, Witlox MA, Stallaert RA, van der Valk P, Postmus PE, Giaccone G. Expression of DNA topoisomerase II{alpha} and topoisomerase IIβ genes predicts survival and response to chemotherapy in patients with small cell lung cancer. Clin Cancer Res 1999;5:2048–58.[Abstract/Free Full Text]
  20. Korkolopoulou P, Angelopoulou M, Siakantaris M, et al. Evaluation of DNA topoisomerase II expression provides independent prognostic information in non-Hodgkin's lymphomas. Histopathology 2001;38:45–53.[CrossRef][Medline]
  21. Schrader C, Meusers P, Brittinger G, et al. Topoisomerase IIalpha expression in mantle cell lymphoma: a marker of cell proliferation and a prognostic factor for clinical outcome. Leukemia 2004;18:1200–6.[CrossRef][Medline]
  22. Korkolopoulou P, Vassilakopoulos TP. Topoisomerase IIalpha as a prognostic factor in mantle cell lymphoma. Leukemia 2004;18:1347–9 (commentary).[CrossRef][Medline]
  23. Provencio M, Corbacho C, Salas C, et al. The topoisomerase II{alpha} expression correlates with survival in patients with advanced Hodgkin's lymphoma. Clin Cancer Res 2003;9:1406–11.[Abstract/Free Full Text]
  24. Vassilakopoulos TP, Doussis-Anagnostopoulou I, Angelopoulou MK, Siakantaris MP, Kittas C, Pangalis GA. Topoisomerase II{alpha} expression in Hodgkin's lymphoma [letter]. Clin Cancer Res 2003;9:5430–1.[Free Full Text]
  25. Stein H, Delsol G, Pileri S, et al. Hodgkin lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. World Health Organization Classification of Tumors: Pathology and Genetics. Tumors of Haematopoietic and Lymphoid Tissues. Lyon; IARC Press; 2001. p. 237–53.
  26. Carbone PP, Kaplan HS, Musshoff K, Smithers DW, Tubiana M. Report of the Committee on Hodgkin's Disease Staging Classification. Cancer Res 1971;31:1860–1.[Free Full Text]
  27. Vassilakopoulos TP, Nadali G, Angelopoulou MK, et al. Serum interleukin-10 levels are an independent prognostic factor for patients with Hodgkin's lymphoma. Haematologica 2001;86:274–81.[Abstract/Free Full Text]
  28. Hasenclever D, Diehl V. A prognostic score for advanced Hodgkin's disease. N Engl J Med 1998;339:1506–14.[Abstract/Free Full Text]
  29. Vassilakopoulos TP, Angelopoulou MK, Siakantaris MP, et al. Prognostic factors in advanced stage Hodgkin's lymphoma: The significance of the number of involved anatomic sites. Eur J Haematol 2001;67:279–88.[CrossRef][Medline]
  30. Vassilakopoulos TP, Nadali G, Angelopoulou MK, et al. The prognostic significance of β2-microglobulin in patients with Hodgkin's lymphoma. Haematologica 2002;87:701–8.[Abstract/Free Full Text]
  31. Canellos GP, Anderson JR, Propert KJ, et al. Chemotherapy of advanced Hodgkin's disease with MOPP, ABVD, or MOPP alternating with ABVD. N Engl J Med 1992;327:1478–84.[Abstract]
  32. Duggan DB, Petroni GR, Johnson JL, et al. Randomized comparison of ABVD and MOPP/ABV hybrid for the treatment of advanced Hodgkin's disease: Report of an Intergroup trial. J Clin Oncol 2003;21:607–14.[Abstract/Free Full Text]
  33. Vassilakopoulos TP, Angelopoulou MK, Siakantaris MP, et al. Combination chemotherapy plus low dose involved field radiation for early clinical stage Hodgkin's lymphoma. Int J Radiat Oncol Biol Phys 2004;59:765–81.[CrossRef][Medline]
  34. Rassidakis GZ, Medeiros LJ, Vassilakopoulos TP, et al. Bcl-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer prognosis in patients treated with ABVD or equivalent regimens. Blood 2002;100:3935–41.[Abstract/Free Full Text]
  35. Angelopoulou MK, Vassilakopoulos TP, Siakantaris MP, et al. EBVD combination chemotherapy plus low dose involved field radiation is a highly effective treatment modality for early stage Hodgkin's disease. Leuk Lymphoma 2000;37:131–43.[Medline]
  36. Doussis-Anagnostopoulou IA, Remadi S, Turley H, et al. Platelet-derived endothelial cell growth factor/thymidine phosphorylase immunohistochemical expression in lymphoid tissue and lymphoid malignancies. Hum Pathol 1997;28:1146–51.[CrossRef][Medline]
  37. Doussis-Anagnostopoulou IA, Garrido MC, Heryet A, et al. Proliferation in Hodgkin's disease: A study using three fixation-resistant markers. Diagn Oncol 1993;3:302–6.
  38. Brown MS, Holden JA, Rahn MP, Perkins SL. Immunohistochemical staining for DNA topoisomerase II{alpha} in Hodgkin's disease. Am J Clin Pathol 1998;109:39–44.[Medline]
  39. Wang J, Taylor CR. Apoptosis and cell cycle-related genes and proteins in classical Hodgkin lymphoma. Application of tissue microarray technique. Appl Immunohistochem Mol Morphol 2003;11:206–13.[Medline]
  40. Kellner U, Heidebrecht HJ, Rudolph P, et al. Detection of human topoisomerase II{alpha} in cell lines and tissues: characterization of five novel monoclonal antibodies. J Histochem Cytochem 1997;45:251–63.[Abstract/Free Full Text]
  41. Sanchez-Aguilera A, Montalban C, de la Cueva P, et al. Tumor microenvironment and mitotic checkpoint are key factors in the outcome of classic Hodgkin lymphoma. Blood 2006;108:662–8.[Abstract/Free Full Text]
  42. Bildriki K, Tel N, Ozalp SS, Yalcin OT, Yilmaz V. Prognostic significance of DNA topoisomerase II-alpha (Ki-S1) immunoexpression in endometrial carcinoma. Eur J Gynaecol Oncol 2002;23:540–4.[Medline]
  43. Watanuki A, Ohwada S, Fukusato T, et al. Prognostic significance of DNA topoisomerase IIalpha expression in human hepatocellular carcinoma. Anticancer Res 2002;22:1113–9.[Medline]
  44. Kreipe H, Alm P, Olsson H, Hauberg M, Fischer L, Parwaresch R. Prognostic significance of a formalin-resistant nuclear proliferation antigen in mammary carcinomas as determined by the monoclonal antibody Ki-S1. Am J Pathol 1993;142:651–7.[Abstract]
  45. Holden JA, Perkins SL, Snow GW, Kjeldsberg CR. Immunohistochemical staining for DNA topoisomerase II in non-Hodgkin's lymphomas. Am J Clin Pathol 1995;104:54–9.[Medline]
  46. Woessner RD, Mattern MR, Mirabelli CK, Johnson RK, Drake FH. Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ 1991;2:209–14.[Abstract]
  47. Kreipe H, Heidebrecht HJ, Hansen S, et al. A new proliferation-associated nuclear antigen detected in paraffin-embedded tissues by the monoclonal antibody Ki-S1. Am J Pathol 1993;142:3–9.[Abstract]
  48. Morente MM, Piris MA, Abraira V, et al. Adverse clinical outcome in Hodgkin's disease is associated with loss of retinoblastoma protein expression, high Ki67 proliferation index, and absence of Epstein-Barr virus-latent membrane protein 1 expression. Blood 1997;90:2429–36.[Abstract/Free Full Text]
  49. Montalban C, Garcia JF, Abraira V, et al. Influence of biologic markers on the outcome of Hodgkin's lymphoma: A study by the Spanish Hodgkin's Lymphoma Study Group. J Clin Oncol 2004;22:1664–73.[Abstract/Free Full Text]
  50. Rassidakis GZ, Medeiros LJ, Drakos E, et al. Low expression of activated caspase-3 (aC3) in Hodgkin and Reed-Sternberg cells (HRS) is independently associated with inferior progression free survival (PFS) in patients with classical Hodgkin's disease (cHD) treated with ABVD or equivalent regimens. Eur J Haematol 2004;73 (Suppl. 65):17 (abstr).
  51. Franklin J, Paulus U, Lieberz D, Breuer K, Tesch H, Diehl V. Is the international prognostic score for advanced stage Hodgkin's disease applicable in early stage patients? Ann Oncol 2000;11:617–23.[Abstract/Free Full Text]
  52. Vassilakopoulos TP, Angelopoulou MK, Siakantaris MP, Kokoris SI, Pangalis GA. Hodgkin's lymphoma: treatment and prognosis. Haema 2004;7:53–63.
  53. Gisselbrecht C, Mounier N, Andre M, et al. How to define intermediate stage in Hodgkin's lymphoma? Eur J Haematol 2005;75:111–4.
  54. Diehl V, Franklin J, Pfreundschuh M, et al. Standard and increased-dose BEACOPP chemotherapy compared with COPP-ABVD for advanced Hodgkin's disease. N Engl J Med 2003;348:2386–95.[Abstract/Free Full Text]
  55. Diehl V, Brillant C, Engert A, et al. Intensification of chemotherapy and concomitant reduction of radiotherapy dose in intermediate stage Hodgkin's lymphoma: results of the fourth interim analysis of the HD11 trial of the GHSG. Eur J Haematol 2004;73 (Suppl. 65):37 (abstr).
  56. Vassilakopoulos TP, Pangalis GA. Biological prognostic factors in Hodgkin's lymphoma. Haema 2004;7:147–64.
  57. Nadali G, Tavecchia L, Zanolin E, et al. Serum level of the soluble form of the CD30 molecule identifies patients with Hodgkin's disease at high risk of unfavorable outcome. Blood 1998;91:3011–16.[Abstract/Free Full Text]
  58. Casasnovas RO, Mounier N, Brice P, et al. Plasma cytokine and soluble receptor signature predicts outcome of patients with classical Hodgkin' lymphoma: a study from the Groupe d' Etude des Lymphomes de l' Adulte. J Clin Oncol 2007;25:1732–40.[Abstract/Free Full Text]
  59. Hochaus S, Di Ruscio A, Di Febo A, et al. Glutathione S-transferase P1 genotype and prognosis in Hodgkin's lymphoma. Clin Cancer Res 2005;11:2175–9.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
B. Chetaille, F. Bertucci, P. Finetti, B. Esterni, A. Stamatoullas, J. M. Picquenot, M. C. Copin, F. Morschhauser, O. Casasnovas, T. Petrella, et al.
Molecular profiling of classical Hodgkin lymphoma tissues uncovers variations in the tumor microenvironment and correlations with EBV infection and outcome
Blood, March 19, 2009; 113(12): 2765 - 3775.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doussis-Anagnostopoulou, I. A.
Right arrow Articles by Pangalis, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doussis-Anagnostopoulou, I. A.
Right arrow Articles by Pangalis, G. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online