Low apoptotic index & bak expression in EBV-associated childhood classical Hodgkin lymphoma.
Background & objectives: Association of Epstein-Barr virus
(EBV) with Hodgkin lymphoma (HL) is particularly high in low-income
countries, and resistance to apoptosis might play a role in pathogenesis
and survival. Data from previous studies are not consistent, and none is
available in children. Thus this study was undertaken on Indian children
with classical Hodgkin lymphoma to assess the significance of bcl-2, bak
and p53 expression, and apoptotic index in relation with EBV status and
treatment outcome with chemotherapy alone.
Methods: Children (age<15 yr) with classical HL (n=143) were included in the study. Bcl-2, bak, p53, Ki67 and latent membrane protein-1 (LMP1) were detected by immunohistochemistry in pre-treatment lymph node biopsies. Apoptotic index was assessed by TdT-dUTP nick-end labelling (TUNEL).
Results: Bcl-2, bak, p53 were expressed above positivity threshold in 83.3, 94.0 and 7.1 per cent of the cases respectively. More than 10 per cent of apoptotic tumour cells were seen in 60.4 per cent of the cases. 131 (91.6%) cases were EBV associated. EBV-positive cases had a significantly lower mean bak expression (p=0.001) and a lower apoptotic index, without higher proliferation index. Advanced stage showed a borderline association with bcl-2 expression in >25 per cent of tumour cells and p53 negative tumours. In univariate analysis, p53 positive cases, which were significantly associated with B symptoms, had a poorer overall survival (P=0.03) while low proliferation index was associated with poorer failure-free survival. Neither EBV status nor any of the apoptotic parameters studied showed independent association with survival.
Interpretation & conclusion: EBV detection in children with classical Hodgkin lymphoma was associated with significant lower bak expression and with lower spontaneous apoptosis of H-RS cells suggesting that EBV-LMP1 might downregulate bak pro-apoptotic protein. This needs to be substantiated further.
Key words Apoptosis--children--Epstein-Barr virus--Hodgkin lymphoma--treatment outcome
|Article Type:||Clinical report|
Epstein-Barr virus (Health aspects)
Epstein-Barr virus (Genetic aspects)
Epstein-Barr virus (Research)
Hodgkin's disease (Risk factors)
Hodgkin's disease (Genetic aspects)
Hodgkin's disease (Research)
Arya, Laxman S.
|Publication:||Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2009 Indian Council of Medical Research ISSN: 0971-5916|
|Issue:||Date: Nov, 2009 Source Volume: 130 Source Issue: 5|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: India Geographic Code: 9INDI India|
Hodgkin lymphoma (HL) is a malignant proliferation of
Reed-Sternberg cells and their variants, Hodgkin cells, in an
inflammatory cellular background, and is characterized by a progressive
painless enlargement of lymph nodes. Its pathogenesis is still poorly
understood. Loss of function of one or more tumour suppressor proteins,
such as p53, may be involved in defective cell regulation of Hodgkin and
Reed-Sternberg (H-RS) cells. p53 detection by immunohistochemistry in HL
is often the result of nonfunctional p53 protein, due to mdm-2 binding,
while p53 mutation are rare. However, over half of paediatric HL cases
have shown that p53 was able to activate its cellular effectors (1).
Control of apoptotic mechanisms seems to play an important role in the pathogenesis of HL and might influence its sensitivity to therapy. Both pro- and antiapoptotic bcl-2 family members, which are important regulators of programmed cell death, are expressed in H-RS cells. Bcl-2 oncoprotein expression was reported in 43-44 per cent of tumour cells from children with HL (1,2). Bak, a bcl-2 antagonist required for p53's ability to induce mitochondrial apoptotic pathway via cytochrome c release from mitochondria (3), is expressed in H-RS cells in 85 per cent of HL tissues (4).
Epstein-Barr virus (EBV) has been detected in H-RS cells in a large subset of HL cases, particularly in developing countries and more among children (5). Inhibition of apoptosis might be especially important in EBV positive cases of HL, as apoptosis is a host cell defense strategy to resist viral infection. In vitro studies have shown that EBV latent membrane protein 1 (LMP1) induces bcl-2 expression in lymphoid cells (6). However, EBV-LMP1 was not found to upregulate bcl 2 expression in HL (2, 7), except in children younger than 6 years (8). No bak regulation by EBV-LMP1 has been shown as yet, either in vitro or in vivo (4).
Since EBV association is higher in paediatric population with HL in India (9-11) as compared with western countries, the expression profile of pro- and anti-apoptotic proteins might also differ. Thus we conducted a study on Indian childhood HL to shed new light on the significance of expression of bcl-2 family members and p53 in relation with clinical and histological factors, EBV association and treatment response.
Material & Methods
Prior approval of the study protocol from the Institute Ethic's Committee was obtained. Of the 178 children with HL seen during the study period, 145 had adequate archival material. Two of these were diagnosed as nodular lymphocyte predominant HL (CD45 and CD20 positive, CD15 and CD30 negative) and were excluded.
Ann Arbor staging: Clinical staging of the patients was performed according to the Cotswolds revision of the Ann Arbor staging system (12). Bulky disease was defined as any tumour with a diameter [greater than or equal to] 6 cm or a mediastinal mass with a diameter exceeding one third of the maximum mediastinal width. Splenic involvement was defined radiologically. Socio-economic status (SES) was assessed by Kuppuswamy's scale (13) and Pareek' scale (14) for patients from urban and rural areas respectively. Moderate and high SES were grouped under high SES. Anaemia was defined as haemoglobin <10.5 g/dl.
Treatment protocol: Most patients were treated with chemotherapy alone, alternating 4 cycles of COPP (cyclophosphamide, vincristine, procarbazine and prednisolone) with 4 cycles of ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine). Eight patients treated in 1990 received 6 to 8 COPP cycles. Additional mediastinal radiotherapy was given to two patients in partial remission (PR) after completing 4 COPP / 4 ABVD.
Treatment response and survival: Patients were assessed for remission status by clinical restaging 4 to 8 wk after completion of chemotherapy. Complete remission (CR) was defined as the complete regression of all clinical, radiological or laboratory evidence of HL. Poor response was defined as failure to achieve CR, i.e., as either PR, or disease progression, or death on therapy. Overall survival (OS) was calculated from diagnosis to death or last visit. Failure-free survival (FFS) was calculated from initial diagnosis to disease- or treatment-related death, failure to attain CR at the end of therapy, disease progression, relapse, whichever came first, or last visit.
Immunohistochemistry: Formalin-fixed paraffin- embedded LN specimens were stained immunohistochemically with primary monoclonal antibodies against CD45, CD20, CD45RO, CD15 and CD30 antigens for immunophenotyping and classifying HL according to the WHO classification (15), bcl-2 (clone 124), p53 (clone DO7), EBV-LMP1 (clone CS 1-4), Ki-67 (clone MIB-1) and with polyclonal rabbit anti bak, as described earlier (11). All primary antibodies were provided by DakoCytomation, Denmark.
The expression of bcl-2, bak, p53 and Ki-67 was analyzed by examining all the fields at a magnifying power of 40X and counting the number of H-RS cellspositive or negative for each immunostain in all fields. Both intensely and weakly stained cells were considered positive. The percentage of H-RS cells expressing each antigen was then calculated. Lymphocyte staining by bcl2, bak and Ki67 was not considered in the quantification of positive H-RS cells but was used as internal control. Proliferation index (PI) was defined as the percentage of Ki-67 expressing H-RS cells. Threshold values for significant protein expression, based on prior studies (16,17), were bcl-2>25 per cent, PI>50 per cent, p53>50 per cent. An arbitrary value of 25 per cent was chosen for significant bak expression, based on a threshold positivity of 10 per cent previously used to define bax positivity in H-RS cells (17), since a similar expression of bak and bax has been described in H-RS cells (4).
Apoptotic index: Apoptotic H-RS cells were detected by TdT-mediated dUTP-Nick-End Labelling (TUNEL) technique using a fluorometric TUNEL system (Promega, USA) as per supplier's instructions. A minimum of 10 fields were examined under a fluorescent microscope (Nikon E 600, USA), selecting the best-stained areas and avoiding areas dominated by sclerosis or fibrosis. Fluorescein-dUTP labelled large cells with a nucleus greater than 2.5 times the size of lymphocyte nuclei were considered as apoptotic HRS cells. Apoptotic index (AI) was calculated as the percentage of fluorescent H-RS cells. A positivity threshold of more than 10 per cent was chosen17, and high AI defined as AI>25 per cent.
Statistical analysis: Statistical analysis was done using SAS 8.0 software. Chi-square test / Fisher's exact test was applied to see significant difference and association between categorical variables. The differences in means of percentage of H-RS cells expressing bcl-2, p53, and AI were compared using student's t-test. Bak expression and PI being non-normally distributed, Mann-Whitney U test was used (18). Binary logistic regression was applied to see the strength of association between categorical factors. Univariate correlation of continuous variables was done by Pearson's correlation coefficient followed by post-hoc analysis. OS and FFS were estimated by Kaplan-Meier actuarial survival method (19). Univariate Cox regression analysis was performed for all candidate prognostic factors. All factors with a log-rank P< 0.10 were included in the multivariate Cox regression model, except for those with no event in one category. All P values were two-sided and a P value < 0.05 was considered as the statistically significant level.
Between December 1990 and January 2004, 128 boys and 15 girls with CHL fulfilled the inclusion criteria. Median age was 8 yr (range 2 to 14) (Table I). Subtype distribution was mixed cellularity in 105 cases, nodular sclerosis in 33, lymphocyte depleted and lymphocyte rich CHL in 1 case each and unclassified CHL in 3 cases.
Apoptosis-related proteins, apoptotic index and proliferation index: Bcl-2 and bak cytoplasmic expression was seen in 110(83.3%) out of 132 and 135(94%) out of 133 cases, with a range of positive expression of 10-65.8 per cent and 10-85.2 per cent of H-RS cells respectively. Nuclear expression of p53, restricted to H-RS cells, was seen in 10(7.1%) out of 140 cases (range 50-70.8%), with a high expression (>75%) in none of the cases. Constitutional symptoms were present in all cases with p53 positive tumours vs. 60.2 per cent of negative tumours (p=0.013). Advanced stage (III and IV) was seen more often in cases with bcl2 >25 per cent (65.5 vs. 51.8%, P=0.1) and p53 positive tumour cells (80.0 vs. 56.2%, P=0.19), although not reaching statistical significance.
AI greater than 10 per cent was observed in 64 (60.4%) out of 106 cases. Bcl-2 expression and AI were poorly correlated (Pearson's correlation coefficient--0.19, P=0.05). All cases showed Ki-67 nuclear staining, with PI greater than 50 per cent in 95 per cent of the cases.
EBV-LMP1 detection: Membrane-bound and cytoplasmic EBV-LMP1 positivity, restricted to HRS cells, was seen in 131 (91.6%) cases and showed no difference between subtypes. EBV-positive cases showed a lower mean bak expression than in EBVnegative cases (27.7 [+ or -] 4.2 vs. 20.7% [+ or -] 9.8, P=0.001). AI was lower in EBV-associated cases, but the difference was not statistically significant (12.9% [+ or -]10.2 vs. 17.0 [+ or -] 9.5). EBV status was unrelated with bcl-2 expression, even in various age subgroups between 5 and 10 yr. p53 expression and PI were not significantly different in EBV positive and negative tumours (Table II).
Response and survival: After exclusion of 12 early defaulters and 9 patients lost to follow up before response assessment, 122 patients were analyzed for response and survival. Median follow up was 4.0 yr (range 0.1-13.7). The ratio of bcl-2 family members rather than any one protein may better determine a cellular response to apoptotic stimuli. Thus the ratio of bcl-2/bak expression in H-RS cells was categorized in various possible ways, but none showed any survival difference between the categories when OS and FFS were assessed (data not shown).
Response to chemotherapy was poorer in cases with p53 expression in more than 50 per cent of H-RS cells [CR in 70.0 vs. 94.5%, P=0.03, unadjusted OR 7.3 (1.535.8)]. P53 expression was associated with poorer OS in univariate analysis (P=0.03, Table III and Fig. 1) but was not independent prognostic factor in multivariate analysis (Table IV). Variables included in Cox regression model were p53 and CD15 expression for OS; stage IV disease, B symptoms, anaemia, p53 and CD15 expression for FFS. Low PI was associated with poorer FFS (P=0.02). However, PI could not be analysed by multivariate analysis, since no events were recorded in the high PI group. CD15 negative tumours and anaemia at presentation were the only independent prognostic factors for poor OS and FFS respectively (Table IV, Figs 2 and 3).
EBV produces two bcl-2 protein homologues, BHRF1 (20) and BALF1 (21). In vitro studies show that bak pro-apoptotic protein binds to BHFR-1, which has an anti-apoptotic function (22). In our paediatric series, EBV positive HL cases had a significantly lower mean bak expression and a lower AI than EBV negative cases. Similar lower expression of bak protein has been shown in EBV associated gastric carcinomas23, while no association has been reported with EBV positive HL4. We therefore suggest that bak downregulation might be one of the mechanisms explaining the lower AI seen in EBV positive cases. Further in vitro studies are required to confirm this hypothesis.
[FIGURE 1 OMITTED]
Bax downregulation and bcl-2 upregulation by EBV may also induce apoptosis resistance. Expression of bax pro-apoptotic protein in HL was previously found to be inversely correlated with EBV24 but was not assessed in the present study. In contrast with in vitro studies, bcl-2 expression was not correlated with EBV-LMP1 in our study population. The potential anti-apoptotic effect of EBV might be regulated by EBV bcl-2 homologues, independently of cellular bcl-2, or by other apoptotic pathways.
The prognostic role of EBV-association is controversial. Some studies on adult patients reported a beneficial effect of EBV on HL survival (9,25,26), others found no significant survival difference, while a lower survival was seen in patients older than 50 yr (27,28). In our paediatric population survival was not significantly different in EBV positive and EBV negative tumours, similar to previous reports (29,30).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
We found that bak was expressed in almost all cases of childhood HL. The level of bak expression in H-RS cells was unrelated to disease stage, treatment response or survival of children with HL. The potential proapoptotic function related to bak might be surpassed by a stronger expression of anti-apoptotic molecules, such as bcl-2, which would explain tumour progression in HL. We report bcl-2 expression (>10% of bcl-2 positive H-RS cells) in 83.3% of childhood HL cases, a much higher figure than previous reports in adults (61%) (31) and in children with HL (43-44%) (1,2).
Previous reports have described an inverse correlation between bcl-2 expression and AI (32,33). These data are consistent with the notion that overexpression of bcl-2 may induce the blockage of apoptosismediated death in HL. High bcl-2 expression in H-RS cells was found to be associated with a poor response to chemotherapy in adults with nodular sclerosis HL (34). Other authors reported a poor OS and FFS in bcl-2 positive CHL cases (29). We also found that cases with moderate and high bcl-2 expression in H-RS cells (>25%) had a poorer FFS, although this association did not reach statistical significance.
In conclusion, EBV detection in Indian children with CHL was significantly associated with lower expression levels of the anti-apoptotic protein bak. A lower pre-treatment AI was also seen in EBV positive cases, although it did not reach statistical significance. These findings need to be substantiated with a prospective study in a larger study population in order to assess whether EBV-LMP1 downregulates bak and diminishes the spontaneous apoptosis of H-RS cells.
Authors acknowledge the Indian Council of Medical Research, New Delhi, India. for financial support and thank Ms Rajani Unni for her technical assistance.
Received March 31, 2008
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Reprint requests: Dr V. Dinand, Pediatric Hematology Oncology, Centre for Child Health, Sir Ganga Ram Hospital, Rajender Nagar New Delhi 110 060, India e-mail: firstname.lastname@example.org
Veronique Dinand, Ajay Malik *, Binimaya Mohanty *, Bal Chander *, Laxman S. Arya ** & Ramesh Dawar ([dagger])
Departments of Pediatrics, * Pathology, All India Institute of Medical Sciences & ** Cancer Institute Indraprastha Apollo Hospital, New Delhi, India
([dagger]) Present address: Dharamshala Cancer Hospital & Research Centre, New Delhi, India
Table I. Clinical findings in 143 children with classical Hodgkin lymphoma Characteristic Study patients Outcome data available N (%) N Sex Male 128 (89.5) 110 (90.2) Female 15 (10.5) 12 (9.8) Age (yr) <5 17 (11.9) 15 (12.3) 5-9 92 (64.3) 79 (64.7) 10-14 34 (23.8) 28 (23.0) Stage I 20 (14.0) 19 (15.6) II 41 (28.7) 35 (28.7) III 64 (44.8) 52 (42.6) IV 18 (12.6) 16 (13.1) B symptoms Absent 53 (37.6) 47 (39.2) Present 88 (62.4) 73 (60.8) Bulky disease Absent 81 (57.0) 68 (56.2) Present 61 (43.0) 53 (43.8) Table II. Relationship between biological markers and EBV status, proliferation and apoptotic index EBV-LMP 1 Neg (%) Pos (%) P Bcl2 [less than or equal to] 25% 7 (8.2) 78 (91.8) 1.0 >25% 5 (8.6) 53 (91.4) Bak [less than or equal to] 25% 3 (3.1) 94 (96.9) 0.001 * >25% 8 (22.2) 28 (77.8) P53 [less than or equal to] 50% 10 (7.7) 120 (92.3) 0.2 * >50% 2 (20.0) 8 (80.0) AI [less than or equal to] 25% 9 (10.0) 81 (90.0) 0.5 * >25% 3 (5.7) 50 (94.3) PI [less than or equal to] 75% 10 (8.8) 103 (92.2) 1.0 * >75% 2 (6.7) 28 (93.3) PI [less than or equal to] 75% (%) >75 % (%) p Bcl2 [less than or equal to] 25% 68 (80.0) 17 (20.0) 0.7 >25% 45 (77.6) 13 (22.4) Bak [less than or equal to] 25% 76 (78.4) 21 (21.6) 0.5 >25% 30 (83.3) 6 (16.7) P53 [less than or equal to] 50% 107 (82.3) 23 (17.7) 0.10 * >50% 6 (60.0) 4 (40.4) AI [less than or equal to] 25% 71 (78.9) 19 (21.1) 1.0 >25% 42 (79.2) 11 (20.8) PI [less than or equal to] 75% -- -- >75% -- -- AI [less than or equal to] 25% (%) >25% (%) P Bcl2 [less than or equal to] 25% 52 (61.2) 33 (38.8) 0.6 >25% 38 (65.5) 20 (34.5) Bak [less than or equal to] 25% 66 (68.0) 31 (32.0) 0.4 >25% 22 (61.1) 14 (38.9) P53 [less than or equal to] 50% 84 (64.6) 46 (35.4) 0.7 * >50% 6 (60.0) 4 (40.0) AI [less than or equal to] 25% -- -- >25% -- -- PI [less than or equal to] 75% 71 (62.8) 42 (37.2) 0.9 >75% 19 (63.3) 11 (36.7) * Fisher's exact test (2-sided). AI, apoptotic index; PI, proliferation index Table III. Overall and failure-free survival in 121 children with classical Hodgkin's lymphoma (univariate analysis) Factors N Death Failure Stage I-II 54 0 7 III-IV 68 9 16 B symptoms A 47 0 4 B 73 9 19 Hepatomegaly No 82 3 8 Yes 37 6 14 Splenomegaly No 67 4 7 Yes 53 5 15 Anemia No 44 0 3 Yes 75 8 18 SES High 34 1 2 Low 72 5 18 CD15 Neg 12 4 6 Pos 109 5 17 EBV Neg 9 1 2 Pos 113 8 21 Bcl-2 [less than or equal to] 25% 71 4 11 >25% 51 5 12 Bak [less than or equal to] 25% 86 6 15 >25% 28 3 7 AI [less than or equal to] 25% 78 5 14 >25% 44 4 9 p53 [less than or equal to] 50% 109 7 20 >50% 10 2 3 PI [less than or equal to] 75% 96 9 23 >75% 26 0 0 Factors Overall survival 5-yr OS P value (SE) Stage I-II 100 0.002 III-IV 85.7 (4.8) B symptoms A 100 0.007 B 87.1 (4.3) Hepatomegaly No 95.4 (2.6) 0.02 Yes 85.4 (6.1) Splenomegaly No 92.9 (3.5) 0.42 Yes 91.3 (4.2) Anemia No 100 0.02 Yes 89.6 (3.8) SES High 97.1 (2.9) 0.35 Low 91.4 (3.8) CD15 Neg 62.3 (16.3) 0.0002 Pos 95.1 (2.1) EBV Neg 88.9 (10.5) 0.64 Pos 92.7 (2.7) Bcl-2 [less than or equal to] 25% 93.6 (3.1) 0.31 >25% 90.6 (4.5) Bak [less than or equal to] 25% 93.3 (2.9) 0.49 >25% 87.4 (6.9) AI [less than or equal to] 25% 92.4 (3.3) 0.63 >25% 92.3 (4.3) p53 [less than or equal to] 50% 93.6 (2.6) 0.03 >50% 77.8 (13.9) PI [less than or equal to] 75% 90.6 (3.2) 0.14 >75% 100 Factors Overall survival HR 95% CI Stage I-II 69.9 n.a. III-IV B symptoms A 55.5 n.a. B Hepatomegaly No 4.6 1.1--18 Yes Splenomegaly No 1.7 0.5--6.3 Yes Anemia No 50.6 n.a. Yes SES High 2.7 0.3--23 Low CD15 Neg 8.1 2.2--30 Pos EBV Neg 0.6 0.1--4.9 Pos Bcl-2 [less than or equal to] 25% 0.5 0.1--1.9 >25% Bak [less than or equal to] 25% 1.6 0.4--6.5 >25% AI [less than or equal to] 25% 26.3 n.a. >25% p53 [less than or equal to] 50% 4.8 1.0--24 >50% PI [less than or equal to] 75% 28.6 n.a. >75% Factors Failure-free survival 5-yr FFS P value (SE) Stage I-II 93.4 (3.7) 0.02 III-IV 74.5 (6.3) B symptoms A 97.3 (2.8) 0.002 B 73.0 (6.1) Hepatomegaly No 91.6 (3.3) 0.0004 Yes 68.2 (8.6) Splenomegaly No 91.8 (3.6) 0.008 Yes 74.5 (6.9) Anemia No 92.0 (4.5) 0.002 Yes 80.0 (5.5) SES High 93.2 (4.7) 0.006 Low 77.9 (5.7) CD15 Neg 55.6 (15.2) 0.001 Pos 86.1 (3.8) EBV Neg 77.8 (13.9) 0.78 Pos 83.8 (4.0) Bcl-2 [less than or equal to] 25% 89.7 (3.7) 0.16 >25% 73.4 (7.5) Bak [less than or equal to] 25% 84.7 (4.3) 0.25 >25% 70.3 (12.5) AI [less than or equal to] 25% 82.7 (4.9) 0.85 >25% 83.9 (6.2) p53 [less than or equal to] 50% 84.6 (3.9) 0.10 >50% 64.0 (17.5) PI [less than or equal to] 75% 79.5 (4.5) 0.02 >75% 100 Factors Failure-free survival HR 95% CI Stage I-II 5.3 1.1--6.8 III-IV B symptoms A 4.7 1.6--14 B Hepatomegaly No 4.3 1.8--10 Yes Splenomegaly No 3.1 1.6--20 Yes Anemia No 5.6 1.6--20 Yes SES High 6.1 4.1--26 Low CD15 Neg 4.1 1.6--10 Pos EBV Neg 1.2 0.3--5.2 Pos Bcl-2 [less than or equal to] 25% 1.8 0.8--4.1 >25% Bak [less than or equal to] 25% 1.7 0.7--4.1 >25% AI [less than or equal to] 25% 0.9 0.3--3.3 >25% p53 [less than or equal to] 50% 2.7 0.8--9.1 >50% PI [less than or equal to] 75% 27.8 n.a. >75% OS, overall survival; HR, unadjusted hazard ratio; CI, confidence interval; FFS, failure-free survival; SE, standard error; n.a., not applicable (no death/event in one of the groups). SES, socio-economic status; AI, apoptotic index; PI, proliferation index. Table IV. Cox multivariate survival analysis for overall and failure-free survival Factors OS FFS Adjusted 95% CI Adjusted 95% CI HR HR Advanced stage n.a. n.a. 1.5 0.50 -4.2 B symptoms n.a. n.a. 2.2 0.6 -7.7 Anaemia n.a. n.a. 3.8 1.0 -14.1 High p53 4.9 0.8 -31.6 2.4 0.6 -9.5 CD15 negative 5.7 1.1 -29.3 2.7 0.9 -8.6 OS, overall survival; FFS, failure-free survival; HR, hazard ratio; CI, confidence interval; n.a., not applicable since no adverse event occurred in one of the groups; PI, proliferation index
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