Topoisomerase IIA Expression As an Independent Prognostic Factor In

Imaging, Diagnosis, Prognosis

To p o i s o m e r a s e II A Expression as an Independent Prognostic Factor in Hodgkin’s Lymphoma Ipatia A. Doussis-Anagnostopoulou,1Theodoros P.Vassilakopoulos,2 Irini Thymara,3 Penelope Korkolopoulou,3 Maria K. Angelopoulou,2 Marina P. Siakantaris,2 Styliani I. Kokoris,2 Evangelia M. Dimitriadou,2 Christina Kalpadakis,2 Marina Matzouranis,2 Loukas Kaklamanis,1 Panayiotis Panayiotidis,2 Marie-Christine Kyrtsonis,2 Athina Androulaki,3 Efstratios Patsouris,3 Christos Kittas,1and Gerassimos A. Pangalis2

Abstract Purpose: To correlate the immunohistochemical expression of topoisomerase IIa (topoIIa)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 (topoIIa) was performed in 238 HL patients. MiB1 (Ki-67) expression was evaluated in 211/238. Results: The mean F SD percentage of topoIIa- and Ki-67^ positive Hodgkin-Reed-Sternberg (HRS)cellswas63F 19% (5%-98%) and 73 F 19% (8%-99%), respectively. The median percentage of topoIIa-positive HRS cells was 64% (interquartile range, 51-78%).There was no correlation between topoIIa expression and patient characteristics.TopoIIa and Ki-67 expression were correlated (Spearman’s Rho 0.255, P < 0.001). TopoIla expression within the highest quartile of this patient population was predictive of failure free survival (FFS) (10-year rates 82 F 3% vs 68 F 7%, P = 0.02 for patients falling into the quartiles 1-3 and 4 respectively). In multivariate analysis topoIIa expression was independently predictive of FFS. Conclusion: To p o II a was expressed in all cases of HL showing a correlation with Ki-67 expression. Under current standard therapy including drugs inhibiting its activity, topoIIa was an independent adverse predictor of FFS with no statistically significant correlation with other established prognostic factors.

Topoisomerases are enzymes that relax DNA supercoiling appear to stabilize the DNA-topoIIa complex and inhibit DNA arising in a variety of nuclear processes. The mechanism of relegation, resulting in an accumulation of lethal double-strand action of topoisomerases I and II involves the formation of DNA breaks (4). Experimental studies have shown that cells transient single or double-stranded DNA breaks respectively. expressing high levels of topoIIa are drug sensitive, while those Topoisomerase II is found in two isoforms, namely top- with low levels are drug resistant (5), making the enzyme a oisomerase IIa (topoIIa) and topoisomerase IIh. TopoIIa is a potentially useful predictor of tumour sensitivity to chemo- target for several cytotoxic agents used in the treatment of therapy. hematologic malignancies, such as doxorubicin, epirubicin, Interestingly, topoIIa is mainly expressed in proliferating mitoxantrone, etoposide, and teniposide (1–3). These agents 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). TopoIIa expression demonstrates a positive correlation with the widely used proliferative marker Authors’ Affiliations: 1Laboratory of Histology and Embryology, 2First Ki67 in a variety of normal and neoplastic tissues (8–11). Department of Internal Medicine and Department of Haematology and The dual role of topoIIa makes it an interesting subject of 3 Department of Pathology, National and Kapodistrian University of Athens, investigation in order to explore the association of its expres- Medical School, Athens, Greece Received 6/7/07; revised 10/2/07; accepted 10/17/07. sion with the outcome of patients with neoplastic disorders. Grant support: IASIS, a nonprofit organization raising funds for research in Thus it is not yet clear whether high levels of topoIIa are leukemias, lymphomas, and related disorders. beneficial, increasing tumor sensitivity to topoIIa inhibitors, or The costs of publication of this article were defrayed in part by the payment of page they are associated with worse prognosis reflecting increased advertisement charges. This article must therefore be hereby marked in accordance proliferative activity. Clinical results in the literature are so far with 18 U.S.C. Section 1734 solely to indicate this fact. a Note: I.A. Doussis-Anagnostopoulou and T.P.Vassilakopoulos contributed equally contradictory. While in some studies high levels of topoII to this work and both should be considered as first authors. have been associated with chemosensitivity, especially in Requests for reprints: Ipatia Doussis-Anagnostopoulou, Laboratory of Histology anthracycline treated breast cancer (12–14), in others topoIIa and Embryology, Athens Medical School, 75, Micras Asias str., 11527 Goudi, expression was associated with inferior outcome (15–19). In Athens, Greece. Phone: 0030-210-7462348/0030-210-6717049; E-mail: a [email protected]. various subtypes of non-Hodgkin’s lymphomas, high topoII / F 2008 American Association for Cancer Research. Ki67 ratio and high topoIIa expression independently pre- doi:10.1158/1078-0432.CCR-07-1395 dicted for inferior outcome (20–22).

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In a recent report on 42 cases of Hodgkin’s lymphoma (HL), without knowledge of patients’ clinical data and outcome. Evaluation low levels of topoIIa expression were associated with shorter of MIB1 (Ki67) expression was also performed without knowledge of a survival (23), but this was not confirmed by a preliminary the topoII status and patients’ clinical data and outcome. study of 57 patients from our group (24). Both these studies The intraobserver and interobserver variability in the evaluation of topoIIa expression was assessed in a subgroup of 28 patients by IADA were small and used different patient selection criteria as well as a and PK. different cuttofs for topoII dichotomization. In view of these Statistical analysis. The mean percentages of topoIIa expressing a conflicting data, we extended our observations on topoII HRS cells among various subgroups of patients were compared by expression in a much larger series of 238 patients with HL, who the Student’s t-test or the one-way analysis of variance (one-way had been homogenously treated with topoIIa inhibitors, ANOVA). The intraobserver and interobserver variability of topoIIa including chemotherapy based on ABVD or equivalent regi- expression was evaluated by the Pearson’s correlation coefficient. The mens with or without radiotherapy. TopoIIa expression was correlation between topoIIa and Ki67 expression was evaluated by correlated with clinicopathological parameters, the expression Spearman’s rho coefficient, given that the distribution of Ki67 of Ki67 and patient outcome. 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 Patients and Methods or partial remission (CR, PR) during initial therapy, requiring switch to another chemotherapy regimen, death during initial therapy, or Patients and staging. We analyzed 238 patients with HL, who were progression after an initial CR or PR. Overall survival (OS) was diagnosed and treated at the Haematology Section, First Department of defined as the time interval between treatment initiation and death Internal Medicine, ‘‘Laikon’’ General Hospital, National and Kapodis- of any cause or last follow-up. Survival curves were plotted by the trian University of Athens. All patients were older than 14 years, were method of Kaplan-Meier. The identification of prognostic factors in HIV-negative, had pretreatment archival lymph node tissue material univariate analysis was based on the log-rank test. Multivariate available for topoisomerase IIa immunostaining and had received analysis was performed using Cox’s proportional hazards model. A treatment with anthracycline-based chemotherapy with or without backward stepwise selection procedure, with entry and removal radiotherapy. Approval was obtained by the appropriate Institutional criteria of P = 0.05 and P = 0.10, respectively, was used. In order to Review Board. All histologic material was reviewed and classified avoid the use of arbitrary cutoffs, we performed survival analysis according to the recent WHO classification (25). The baseline patient according to the quartiles of topoIIa expression. High topoIIa characteristics, median follow-up, and failure free survival rates were expression was defined as percentages of topoIIa-positive HRS cells comparable with those of patients who had also received anthracycline- falling into the upper quartile (designated as Q4) vs. those falling based chemotherapy with or without radiotherapy during the same within the three lower ones (designated as Q1-3). TopoIIa expression period, but had not tissue material available for topoisomerase IIa was also evaluated as a continuous covariate (percentage of topoIIa- immunostaining (all P-values >0.05; data not shown). positive HRS cells in each case) in multivariate analysis. Furthermore, All patients were clinically staged according to the Ann-Arbor system topoIIa expression was evaluated according to the cutoff obtained by (26), using standard staging procedures. Clinical stages IA and IIA´ were a ROC curve-based approach. Finally, topoIIa expression was tested considered early, while clinical stages IB, IIB, III and IV were considered against the IPS value in multivariate analysis (28); all individual IPS advanced. Anemia was defined as the presence of hemoglobin levels variables were excluded from this analysis. <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 Â 109/l or <8% respectively (28). Results The number of involved anatomic sites was determined as previously described (29). Patients’ characteristics Treatment strategies. Treatment strategies for early Ann-Arbor stage Patients’ clinical and laboratory characteristics are summa- (AAS IA, IIA) and advanced stage (IB, IIB, III, IV) patients have been rized in Table 1. They were compatible with other reported described previously (30). Early stage patients were scheduled for unselected series of patients with non-pediatric HL. The median combined modality therapy including low-dose involved field radio- age of the patients was 30 years (15-82) and 117 (49%) were therapy. Advanced stage patients received chemotherapy. Radiotherapy males. Histologically, there were 232 cases of classical HL was administered to 68% of patients with AAS IB, IIB and IIIA. In (97%) and 6 cases of nodular lymphocyte predominance contrast only 26% of patients with AAS IIIB and IV received radiotherapy. ABVD or EBVD was administered to 82% of the patients, (NLP) HL (3%). In detail, 174 patients were classified as alternating MOPP/ABVD or MOPP/EBVD to 12% and MOPP/ABV or nodular sclerosis (NS) subtype (73%), 47 cases as mixed MOPP/EBV hybrid to 6%. All these regimens are currently considered cellularity (MC) (20%), 8 as lymphocyte rich (3%), 1 as equivalent (31–35). lymphocyte depletion (<1%), 1 as interfollicular (<1%) and 1 Immunohistochemical staining. Immunohisthochemical staining as classical HL, unclassified (<1%). The median follow-up of was performed as previously described (36). The monoclonal patients, who were alive at the time of the analysis, was 111 antibodies Ki-S1 (DAKO, Denmark; dilution 1:50), which recognises months (23-222). the topoIIa isophorm and MIB1 (YLEM; dilution 1:100) for Ki67 were used. Omission of the primary antibody was used as a negative Evaluation of topoIIa and Ki67 expression control in all cases, while a reactive lymph node was used as a positive Immunohistochemical expression of topoIIa was seen in all control. cases and was mainly present in the neoplastic HRS cells and Nuclei from at least 100 neoplastic Hodgkin Reed-Sternberg (HRS) variants. A number of reactive small lymphocytes and larger cells were to be evaluated in each case. This was possible in approximately 90% of the cases, while in the remaining cases we cells within residual germinal centers were also positive. The evaluated as many HRS cells as possible (usually >80). The labelling staining observed was diffuse nuclear (Fig. 1A), with an index (LI) was calculated as the percentage of positive cells. All nuclear occasional punctate pattern. The percentage of positive HRS staining, whether weak or strong, was counted as positive. The cells ranged from 5% to 98%, following an approximately evaluation of topoIIa expression was blinded, since it was performed normal distribution with a mean F SD of 63 F 19% and a

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median value of 64% (interquartile range, 51-78%). Both gender, where males had marginally higher expression than intraobserver and interobserver variability were very low with women (P = 0.08; Table 1). Although the 6 patients with NLP Pearson’s correlation coefficients of 0.943 (P < 0.001) and had somewhat higher mean percentage of topoIIa expression 0.950 (P < 0.001) respectively. than patients with classical HL (75 F 17% vs. 63 F 19%), the No statistically significant relationship was found between difference was not statistically significant (P = 0.16). TopoIIa topoIIa expression and demographic, clinical, and convention- expression did not also differ according to the subtypes of al laboratory parameters including IPS, with the exception of classical HL (Table 1).

Table 1. Correlation of the percentage of topoisomerase IIa-positive HRS cells with baseline characteristics of patients with Hodgkin’s lymphoma

Patients’ Characteristics Patients Topo IIA (mean F SD, %) No. % Age (years; n = 238) <45 182 76 63 F 19 z45 56 24 66 F 20 Sex (n = 238) male 117 49 66 F 19* female 121 51 61 F 20* Histologyc (n = 238) NLPc 63 75F 17 LRc 83 59F 27 NSc 174 73 63 F 19 MCc 47 20 65 F 19 B-symptoms (n = 238) no 152 64 63 F 20 yes 86 36 64 F 19 Ann Arbor Clinical Stage (n = 238) I542367F 19 II 116 49 62 F 20 III 40 17 63 F 19 IV 28 12 65 F 19 # Involved Anatomic Sites (n = 238) V4 201 84 64 F 19 z5371662F 22 Anemia (n = 232) no 143 62 63 F 19 yes 89 38 64 F 20 White blood cells (n = 233) <15 Â 109/l 196 84 64 F 19 z15 Â 109/l 37 16 59 F 23 Severe lymphopenia (IPS cutoff; n = 204) no 190 93 64 F 20 yes 14 7 70 F 17 ESR (mm the 1st hour; n = 183) <50 95 52 66 F 19 z50 88 48 61 F 21 Serum Albumin (g/dl; n = 189) z4 120 63 65 F 19 <4 69 37 66 F 19 LDH levels (n = 188) normal 128 68 64 F 20 elevated 60 32 64 F 18 International Prognostic Score (n = 165)b 0261565F 21 1653964F 16 2392373F 19 3231461F 21 411763F 24 z54275F 3

NOTE: The number of patients with available values for each parameter is denoted by n (in parentheses). Abbreviations: NLP, nodular lymphocyte predominance; NS, nodular sclerosis; MC, mixed cellularity; LR, lymphocyte rich classical Hodgkin’s lymphoma. *Non-significant results for all comparisons (all P values >0.10), except of gender (borderline P value, 0.08). cNLP vs classical HL: P = 0.16. Three additional patients had lymphocyte depletion (n = 1), interfollicular HL (n = 1) and classical HL, unclassified (n = 1). bIndividual value International Prognostic Score (IPS) comparison was based on 168 patients, but it was possible to determine whether IPS were <3 or z3 in 210 patients. TopoIIa expression did not differ in the latter groups as well.

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Fig. 1. A, topoisomerase IIa 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 IIa 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).

Ki67 expression was evaluated in 211/238 patients (89%). established conventional prognostic factors proved to be The staining was diffuse nuclear and the mean F SD percentage statistically significant. Except of quartile analysis, a ROC of positive HRS cells was 73 F 19% (range 8%-99%). The curve-based approach was used to dichotomize topoIIa values. median Ki67 expression was 76% (interquartile range, 61-88%). According to the latter a cutoff of 74.8% was suggested, which A rather loose positive correlation was found between TopoIIa was rounded to 75%. and Ki67 expression (Spearman’s rho 0.255, P < 0.001). The 10-year FFS was 82 F 3% vs 68 F 7% for patients with topoIIa expression within Q1-3 and Q4, respectively Univariate analysis (P = 0.02) (Fig. 1B and Table 2). Using 75% as ROC-based Remission rates. Remission rates did not differ according to cutoff for topoIIa expression, the discrimination was even topoIIa expression: Primary refractory disease was observed in better with 10-year FFS being 84 F 3% vs 67 F 6% (P = 0.002). 4.5% vs. 3.5% of patients with topoIIa expression within Q1-3 In this analysis 72 patients (30%) fell into the adverse group. and Q4 respectively (P = 0.73). Furthermore, early failure, Failure-free survival in stage and IPS subgroups. Among 127 defined as progression or relapse within the 1st year from diag- patients with early stage HL (IA/IIA), topoIIa expression did not nosis, was observed in 7.8% vs. 5.1% of patients respectively show a statistically significant association with FFS (10-year rates (P = 0.48). 89 F 3% vs 83 F 7%, P = 0.36 for patients with topoIIa Failure-free survival in all patients. The results of univariate expression within Q1-3 and Q4 respectively; Fig. 1C). In analysis of FFS are summarized in Table 2. Most of the contrast, topoIIa expression was significantly associated with

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

Prognostic factor 10-y FFS (%) P Topoisomerase IIa expression (Q1-3 vs Q4)* 82 F 3vs68F 7 0.02 Topoisomerase IIa expression (< vs z75%) 84 F 3vs67F 6 0.002 Age (<45 vs z45 y) 81 F 3vs73F 6 0.14 Sex (male vs female) 78 F 4vs80F 4 0.76 Histology (NLP vs LR vs NS vs MC)c 67 F 19 vs 100 vs 77 F 3vs84F 6 0.60 B symptoms (no vs yes) 85 F 3vs69F 5 0.002 Ann Arbor Clinical Stage (I vs II vs III vs IV) 90 F 4vs80F 4vs76F 7vs57F 10 0.008 Ann Arbor Clinical Stage (I/II/III vs IV) 82 F 3vs57F 10 0.004 # Involved anatomic sites (V4vsz5) 82 F 3vs60F 9 0.002 Anemia (no vs yes) 82 F 3vs72F 5 0.07 White blood cells (< vs z15 Â 109/l) 80 F 3vs72F 8 0.32 Severe lymphopenia (IPS cutoff; no vs yes) 79 F 3vs64F 13 0.11 ESR (< vs z50 the 1st hour) 85 F 4vs69F 5 0.001 Serum albumin (z vs < 4 g/dl) 84 F 4vs68F 6 0.0007 LDH levels (normal vs elevated) 79 F 4vs76F 6 0.76 International Prognostic Score (<3 vs z3) 81 F 3vs64F 8 0.002

NOTE: Percentages represent 10-y failure-free survival (FFS) rates F standard error. *Q1-3 = quartiles 1, 2, and 3 (lower); Q4 = quartile 4 (upper). cNLP vs classical HL: 67 F 19 vs 79 F 3; P = 0.47.

FFS among the 111 patients with advanced disease (10-year the contrary, the effect of high topoIIa expression in patients rates 74 F 5% vs 52 F 11%, P = 0.03 for patients with expression treated with combined modality was much smaller: 10-year FFS within Q1-3 and Q4; Fig. 1D). When patients were stratified rates were 88 F 3% vs. 82 F 6% in patients falling within Q1-3 according to the IPS, topoIIa expression had again a non- vs. Q4 respectively (P = 0.25). Among 169 patients treated with significant effect on FFS in patients with IPS <3 [10-year rates combined modality, 114 (67%) had early stage disease. 82 F 4% vs 76 F 8%, P = 0.64 for patients with expression Overall survival. During follow-up 33 deaths were recorded: within Q1-3 and Q4 respectively], and a highly significant effect 20 (61%) due to HL-related and 13 (39%) due to unrelated on FFS of patients with IPS z3 [corresponding 10-year rates causes. 74 F 8% vs 30 F 14%, P = 0.0008], so that patients with IPS z3 The difference in overall survival was borderline: Patients and high topoIIa expression had a particularly poor outcome. with topoIIa expression within Q1-3 had a 88 F 3% 10-year Failure-free survival in treatment modality subgroups. High overall survival rate vs. 81 F 5% for those with expression topoIIa expression was highly predictive of adverse outcome within Q4 (P = 0.06). At the 75% ROC-based cutoff the overall in patients treated with chemotherapy only; 10-year FFS rates survival difference was significant (10-year rates 90 F 3% vs. were 71 F 7% vs. 29 F 15% in patients falling within Q1-3 vs. 79 F 5%, P = 0.02).Patients with topoIIa expression within Q4 respectively (P = 0.003). Among 69 patients who received Q1-3 had a 94 F 2% 10-year cause-specific survival rate vs 83 F chemotherapy alone, 56 (81%) had advanced stage disease. On 5% for those with topoIIa expression within Q4 (P = 0.005).

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

Prognostic Factor Relative risk Exp(b) 95% CI P Model 1: Covariates with >10% missing values excluded (238 patients) TopoIIa (Q4 vs Q1-3) 2.0 1.1-3.6 0.02 B-symptoms (yes vs no) 2.0 1.1-3.7 0.02 No. involved anatomic sites (z5vsV4) 1.9 0.99-3.77 0.06

Model 2: Covariates with >10% missing values included (189 patients) Serum albumin (<4 vs z4 g/dl) 2.6 1.3-4.9 0.005 TopoIIa (Q4 vs Q1-3) 2.3 1.2-4.5 0.01 Ann Arbor Clinical Stage (IV vs I/II/III) 2.4 1.1-5.1 0.03

Model 3: Only topoisomerase IIa and IPS included (210 patients) IPS (z3 vs <3) 2.8 1.5-3.8 0.001 TopoIIa (Q4 vs Q1-3) 2.0 1.1-3.8 0.02

NOTE: Topoisomerase IIa expression was analyzed at the cutoff of the upper quartile (Q4) vs the three lower ones (Q1-3). Abbreviations: 95% CI = 95% confidence intervals.

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The discriminative ability at the 75% cutoff was even better (10- neoplastic cells in 15 cases of LPHL was 84% (38). This result year rates 96 F 2% vs. 80 F 5%, P = 0.0003). was obtained from the evaluation of CD20/topoIIa double positive cells. Although the mean percentage of topoIIa- Multivariate analysis positive neoplastic cells in our NLP HL patients was numeri- TopoIIa expression was evaluated together with covariates, cally higher than the one observed in classical HL, the which were significant in univariate analysis and had <10% difference did not reach statistical significance. However the missing values, i.e. stage IV, B-symptoms, and number of number of cases of NLP HL was low to permit a reliable involved anatomic sites. The results of multivariate FFS statistical analysis. In contrast to the high topoIIa expression a analysis are summarized in Table 3. High topoII expression observed in the present, as well as other series (37, 38), other (within Q4) had independent prognostic significance for FFS, investigators have reported lower median percentages of posi- along with B-symptoms and, marginally, the number of tive HRS cells in the order of 30% (23, 39). These differences involved anatomic sites. When covariates with >10% missing may be related to the use of different monoclonal antibodies a values were included, topoII expression within Q4, stage IV, for topoIIa detection, as it has been demonstrated that KiS1 is and reduced serum albumin levels remained independent an antibody with high binding affinity to topoIIa (40). prognostic factors for FFS. We demonstrated here that high topoIIa expression had a When the percentage of topoII -positive HRS cells was independent adverse prognostic significance in a series of 238 evaluated as a continuous covariate, its independent prognostic patients with HL, who had been treated with ABVD or a significance for FFS persisted: Increasing topoII expression equivalent regimens with or without RT in a single center. This (P = 0.02; relative risk 1.020 per percentage unit; 95% CI 1.004- treatment approach invariably included the administration of 1.036), B-symptoms and the number of involved anatomic sites doxorubicine or epirubicine, which exert their antineoplastic were again selected in the multivariate model. action through topoIIa inhibition. Our results contradict those a High topoII expression (within Q4) remained an indepen- reported by Provencio et al. (23), who had suggested a dent prognostic factor for FFS after adjustment for the IPS (P = favorable prognostic impact of higher topoIIa expression in a a 0.02; Table 3). The same was true when topoII expression was series of 42 patients with advanced HL. However, a more recent evaluated as a continuous covariate (P = 0.03; relative risk 1.019 report by the Spanish group demonstrated that high topoIIa per percentage unit; 95% CI 1.002-1.036). When B-symptoms expression (within the corresponding Q4) was associated with and the number of involved anatomic sites were evaluated along inferior cause-specific survival in a series of 235 patients, a a with topoII and IPS, the significance of topoII persisted, while eventhough the difference did not reach statistical significance that of IPS and B-symptoms were borderline. (41). The absolute difference in long-term cause-specific a When topoII expression was analyzed at the ROC-based survival between patients with topoIIa expression within cutoff of 75%, its significance was strengthened in all Q1-3 and Q4 in the latter study was approximately 8-10% multivariate models. (approximation obtained from survival curves), a figure a In multivariate analysis of overall survival topoII had a non- consistent with our data. Our results are also in agreement significant effect after adjustment for age and the number of with the adverse significance of topoIIa expression in other involved sites (P = 0.13). neoplasms (15, 16, 20–22, 42–44), including non-Hodgkin’s lymphomas (20–22), even after chemotherapy with topoIIa Prognostic significance of Ki67 and TopoIIa/Ki67 ratio inhibitors (18–20). In the population of 211 patients with available data on Ki67 A simplified explanation of our findings could be that expression (with 43 failure events), high topoIIa expression topoIIa exerted its effect on the prognosis of HL merely through (within Q4) was again associated with inferior FFS (P = 0.03). its role as a proliferation marker, which is further supported by Irrespectively of the cutoff value used, Ki67 expression was not the positive correlation that we found both between topoIIa associated with FFS. In contrast, the ratio topoIIa/Ki67 was of and MIB1 (Ki-67) expression. The mean percentage of Ki67- prognostic significance: The 10-year FFS was 82 F 3% for the positive cells was higher than that of TopoIIa positive cells, a 147 (70%) patients with ratios <1 vs. 72 F 6% for the 64 (30%) finding described both in our previous study (37) and in non- patients with ratios z1(P = 0.03). In multivariate analysis, a Hodgkin’s lymphomas (20, 45). However topoIIa might topoIIa/Ki67 ratio z1 was independently associated with FFS provide a better estimate of the number of cycling cells than (P = 0.04) along with B-symptoms (P = 0.03) and, marginally, Ki67, because it is selectively present during the later phases of stage IV (P = 0.06). However, neither Ki67 nor the ratio the cell cycle (40, 46, 47). Thus the data presented here are topoIIa/Ki67 added independent prognostic information in compatible with a role of topoIIa as a proliferation marker. multivariate models already including the percentage of In contrast to topoIIa, Ki-67 expression was of no prognostic topoIIa expression. significance in our series. Although Ki67 expression was Discussion predictive of the outcome in the initial study of the Spanish group (48), this was not confirmed in a subsequent larger series The expression of topoIIa in human neoplasia has been the evaluated by tissue arrays (49). Additionally, in accordance subject of many recent reports. In this study all cases of HL with a previously report of our group on non-Hodgkin’s expressed topoIIa with a median number of positive HRS cells lymphomas (20), a ratio of topoIIa/Ki67 z1 was an of 64%. These results are in accordance with those previously independent predictor of inferior FFS in this series as well, described by us in a different patient series (37). Brown et al., in but did not add prognostic information independent of series of 49 cases of HL, found similar percentages of topoIIa- topoIIa expression per se. positive neoplastic cells in cases of classical HL (58% in NS and Except of a borderline association with male gender, topoIIa 68% in MC), while the percentage of topoIIa-positive expression was neither correlated with any of the examined

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This is in contrast simplified approach could suggest that if more topoIIa is with most conventional prognostic factors, which present expressed in the HRS cells, the target for anthracyclines would extensive interrelationships (28), and resembles to what has be more abundant and the antitumor activity would be greater. been observed with a small number of recently described This view has been supported by recent results, especially in biological markers, such as bcl-2 (34) and activated caspase-3 breast cancer, but in others tumors as well (12–14, 23). expression (50). However, as more HRS cells express topoIIa, the amount of Given the lack of correlation with other prognostic factors anthracyclines delivered by standard chemotherapy regimens and its prognostic significance in univariate analysis of FFS, may not suffice to substantially inhibit topoIIa activity and lead topoIIa emerged as an independent prognostic marker in the neoplastic cells to apoptosis. This hypothesis offers an multivariate analysis. Using the upper quartile as a definition additional explanation of our findings. Furthermore, if this is for high topoIIa expression we avoided ’‘data fitting’’, which indeed the major mechanism responsible for the higher failure could arise by the introduction of arbitrary or ‘‘best’’ cutoffs. rates found in patients with higher topoIIa expression, then it The validity of our findings is strengthened by the fact that might have therapeutic implications: it would be logical to topoIIa was not only predictive of FFS at the cut-off of the hypothesize that patients with high levels of topoIIa might upper quartile, but also carried independent prognostic benefit from dose intensification of topoIIa inhibitors or from significance when evaluated as a continuous covariate. A the addition of a second topoIIa inhibitor in the initial ROC-curve defined cutoff (75%) led to the demonstration of chemotherapy regimen. This is the case with BEACOPP- an even stronger prognostic impact of high topoIIa expression. escalated, the most effective but also toxic regimen available Despite its clear independent prognostic significance, we for advanced HL (54), which is now also being tested in further tested whether topoIIa added to the prediction achieved patients with early stages and unfavorable prognostic profile by the IPS, which is the most popular prognostic index for (55). BEACOPP-escalated maintains the dose intensity of advanced HL (28), while very similar systems may also work in doxorubicine in comparison with ABVD, but—in addition— localized disease (51–53). Thus in another multivariate includes 3 days of etoposide, another topoIIa inhibitor, at high approach including both IPS and topoIIa, the latter proved dose of 200 mg/m2 daily. It would be very interesting to see again to be an independent prognostic factor for FFS. whether the prognostic significance of topoIIa expression will The adverse prognostic effect of high topoIIa expression was be overcome by the administration of BEACOPP-escalated, a mainly restricted to advanced stage patients, while its effect in situation that would strengthen our hypothesis. early stages was not statistically significant. Furthermore, high In conclusion, we demonstrated the adverse prognostic topoIIa expression was predictive of adverse outcome in significance of topoIIa expression in patients with HL treated patients treated with chemotherapy only, a finding suggesting with ABVD or equivalent regimens with or without radio- that high topoIIa expression did not confer sensitivity to therapy. Apart from circulating factors including cytokines anthracycline-based chemotherapy. However, since 81% of (27, 56–58), molecules involved in apoptosis (34, 49, 56) patients treated with chemotherapy alone had advanced stage and polymorphisms implicated in drug metabolism (59), disease, it is not clear that the highly significant difference was topoIIa expression appears to have also a place in the arma- related to the applied treatment or to the inclusion of advanced mentarium of biological prognostic factors in HL due to its stage patients. On the contrary, the effect of high topoIIa implication in cell proliferation and drug sensitivity mecha- expression in patients treated with combined modality was nisms. Following appropriate validation, this molecule may much smaller, probably due to the fact that most patients offer a biological rational for treatment intensification with treated with combined modality had early stage disease, topoIIa inhibitors, generating an hypothesis that could be resulting to a low number of failure events, which reduced tested in future trials.

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Ipatia A. Doussis-Anagnostopoulou, Theodoros P. Vassilakopoulos, Irini Thymara, et al.

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