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repp86 Expression and Outcome in Patients With Neuroblastoma

Matthias Krams, Hans-Juergen Heidebrecht, Barbara Hero, Frank Berthold, Dieter Harms, Reza Parwaresch, Pierre Rudolph

From the Department of Pathology, University of Kiel; and German Neuroblastoma Study Group, Department of Pediatrics, University of Cologne, Germany.

Address reprint requests to Pierre Rudolph, MD, PhD, Department of Pathology, University of Kiel, Michaelisstr 11, 24105 Kiel, Germany; email: prudolph{at}path.uni-kiel.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: Given the well-known challenges of neuroblastoma prognosis, we investigated whether the expression of restrictedly expressed proliferation-associated protein of 86 kDa theoretical molecular mass (repp86), a proliferation-associated protein expressed in S, G₂, and M phases of the cell cycle, correlates with the clinical outcome in patients with neuroblastoma.

Patients and Methods: 161 children with different stages of neuroblastoma were studied; the median follow-up time was 72.8 months. The patients were staged according to the International Neuroblastoma Staging System, and histologic grading of the tumors was performed according to the criteria of Hughes and those of the International Neuroblastoma Pathology Classification. The MYCN gene copy number was determined by Southern blot analysis or fluorescence in situ-hybridization, and repp86 expression was assessed immunohistochemically by means of monoclonal antibody Ki-S2 on paraffin sections from archival tumor samples.

Results: A repp86 labeling index (RI) of more than 10% positive tumor cells significantly predicted a shortened disease-free interval and an increased tumor mortality (both P < .0001). Moreover, the RI allowed the identification of patients with favorable and adverse prognosis in subsets defined by stage, grade, age, and MYCN status. In a multivariate analysis, the RI emerged as the most important predictor of event-free and disease-specific survival with hazard ratios of 11.7 and 10.5, respectively (both P < .0001).

Conclusion: It seems that repp86 expression is closely associated with the biologic behavior of neuroblastoma. Assessment of the RI might, therefore, considerably refine prognostic models.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
NEUROBLASTOMA IS the most common malignant solid extracranial tumor in children and accounts for approximately 15% of childhood cancer deaths. Prognostic predictions chiefly rely on two well-established clinical criteria: the extent of the disease (clinical stage) and the patient’s age at diagnosis. However, the behavior of neuroblastoma is eminently capricious and embraces a spectrum ranging from high aggressiveness with early widespread dissemination to spontaneous maturation and even terminal differentiation to benign ganglioneuroma.¹ Because of this biologic heterogeneity, unexpected outcomes are encountered in different stage and age groups, posing a serious problem for the selection of risk-adapted therapy designs.

In addition to histopathologic grading systems with and without adjustment for patient age,^2–6 molecular analyses have been solicited to improve prognostic models. Thus, amplification of the MYCN gene and loss of heterozygosity on 1p32–36, which are likely to affect a common pathogenetic pathway,⁷ are valued for their ability to identify high-risk patients in almost all age and stage groups.^8–12 It nevertheless seems that the MYCN status cannot fully explain the evolution of individual tumors,^13–15 indicating an involvement of additional genetic alterations. Accordingly, recent cytogenetic studies have shown that gain of genetic material on 17q¹⁵ and deletion of 11q23¹⁶ are associated with an adverse outcome even in neuroblastomas with a single MYCN gene copy.

Recently, telomerase activity (TA) was found to carry outstanding prognostic significance independent of tumor stage and patient age.¹⁷ As a mechanism granting unlimited cellular proliferation, TA has been implicated in the development and progression of various cancer types including neuroblastoma.^17–21 Indeed, downregulation of TA may account for the spontaneous regression observed in a subset of neuroblastomas.²² However, the assessment of TA is not likely to become a standard procedure because it requires fresh or well-preserved snap-frozen tissue samples that are not always available to the pathologist. In addition, evaluation of subunits of the telomerase protein complex such as human telomerase and human telomerase reverse transcriptase by means of reverse transcriptase polymerase chain reaction (RT-PCR) or immunohistochemistry does not seem to provide equivalent information,¹⁷ thwarting the routine application of telomerase measurements.

Mindful of the fact that TA is invariably associated with cell proliferation,²³ we studied the expression of restrictedly expressed proliferation-associated protein of 86 kDa theoretical molecular mass (repp86) in neuroblastomas. repp86 was originally identified as a protein migrating to 100 kDa because of abundant phosphorylation in asynchronously cycling cells.²⁴ By cloning of the cDNA,²⁵ repp86 was characterized as the human homolog of the Xenopus protein TPX2,²⁶ which is required for chromatin-induced microtubule assembly during mitosis.²⁷ In mammals, repp86 expression is cell cycle regulated, with a diffuse nuclear distribution becoming apparent at the onset of S phase, persisting through G₂ phase, and relocating to the mitotic spindle in M phase to vanish with the completion of cytokinesis.^24,28 In a recent investigation, we showed that repp86 is required for the formation of a functional mitotic spindle and that its scheduled expression is rate-limiting for mitotic cell division (Steffen R, Parwaresch R, Rudolph P, et al, submitted for publication). These properties together with the availability of a specific monoclonal antibody suitable for paraffin-embedded material, Ki-S2,²⁸ make repp86 the prototype of a new generation of proliferation markers.

In this retrospective study of 161 neuroblastoma patients with long-term follow-up, we show that the prognostic information derived from repp86 expression is more specific than that provided by histopathologic grading, MYCN analysis, and even tumor stage. This exceptional prognostic effect is likely to be attributable to two distinct properties of repp86: a function indispensable for cell division and a close association with both cell cycle deregulation and TA.^29–31

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
The original study group comprised 181 patients enrolled in the German Neuroblastoma Trial who were diagnosed between 1983 and 1999. Of these, 14 were lost to follow-up, and the available data were incomplete for six additional patients. The remaining 161 patients were included in this study with informed consent; 78 patients were males and 83 patients were females. Their median age at diagnosis was 15 months (range, 0 to 191 months). The clinical stage of the disease was evaluated according to the International Neuroblastoma Staging System (INSS),³² with the following results: 16 patients (10%) were in stage 1; 21 patients (13%) were in stage 2; 41 patients (25%) were in stage 3; 55 patients (34.5%) were in stage 4, and 28 patients (17.5%) were in stage 4S (special). The patients in the final stage were markedly younger at the time of diagnosis (median age, 1.5 months) than those in advanced and even localized stages (all P < .0001), whereas the age difference between localized and advanced stages was not significant.

Patients were treated according to the guidelines established by the German Neuroblastoma Trial (NB85, NB90, NB95-S, and NB97), with stratification criteria as described elsewhere.³³ Forty-four of the original 181 patients had been treated with chemotherapy before biopsy; they accounted for 29 of the 161 patients with complete data including follow-up. The median follow-up period was 72.8 months (range, 0.2 to 226.1 months).

Hughes criteria² and the International Neuroblastoma Pathology Classification (INPC)⁶ were employed for histologic typing and grading. Forty-eight tumors (30%) were Hughes grade 1, 52 tumors (32%) were grade 2, and 61 tumors (38%) were grade 3. Grade 1 was further subdivided into diffuse ganglioneuroblastomas (GNBL; 1a; n = 38) and composite GNBL (1b; n = 10), as recommended.^34,35 Because the INPC system is applicable exclusively to biopsy specimens taken before treatment, it could only be employed in 132 patients. These comprised 103 neuroblastomas (32 undifferentiated, 33 poorly differentiated, and 38 differentiating) and 29 GNBL (seven nodular and 22 intermixed). Of these, 69 patients (52.3%) had favorable and 63 patients (47.7%) had unfavorable histology.

Assessment of Tumor Cell Proliferation
Tumor cell proliferation was analyzed immunohistochemically with the monoclonal antibody Ki-S2²⁸ directed against the mitotic spindle-associated protein repp86.^24,25 Briefly, 3- to 5-µm-thick sections were cut from formalin-fixed, paraffin-embedded tumor specimens, mounted on silanized slides, and dried overnight at 37°C. Immunoreactivity was restored by microwave pretreatment in 0.01 M citric acid, pH 6.0, for 30 minutes at high power setting (800 W). The sections were incubated with the primary antibody (undiluted hybridoma cell culture supernatant) for 30 minutes at room temperature, and the staining reaction was enhanced by means of the alkaline phosphatase-antialkaline phosphatase technique.³⁶ Tissue structures were visualized by counterstaining with Mayer’s hematoxylin.

Ki-S2 immunolabeling was evaluated by counting 1,000 tumor cells at high magnification (x400) using a standard light microscope (Zeiss, Oberkochen, Germany) and a hematologic cell counter (Statitest L10, Ferrari, Berlin, Germany), starting from the most densely labeled area of the tumor section. Only manifest nuclear staining was accepted as a positive reaction. Counting was done by two independent observers (M.K. and P.R.); the overall interobserver agreement was 0.94 (Kendall’s tau). The Ki-S2 labeling index (repp86 index [RI]) was calculated as the percentage of positive tumor cell nuclei and rounded off to the nearest integer.

MYCN Amplification Analysis
MYCN copy number was assessed by Southern blot analysis of Escherichia coli restriction enzyme I–digested tumor DNA and hybridization with a probe complementary to the MYCN gene (AppligeneOncor, Illkirch, France), as has been described.³⁷ A small number of cases were investigated by fluorescence in situ hybridization on paraffin-embedded specimens.³⁸ Briefly, paraffin-embedded tumor samples were cut as for immunohistochemistry. After deparaffinization and rehydration, tissue slides were treated with 40 µg/mL proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) in 50 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA at 37°C for 30 minutes. Tissue sections were postfixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 minutes and finally washed in PBS. Ten microliters of hybridization solution containing 10 ng of probe cocktail (AppligeneOncor) was applied to each section and covered with a glass coverslip. After a denaturation period of 5 minutes at 95°C, hybridization was allowed to take place at 37°C for up to 16 hours. After hybridization, the slides were washed as described by the supplier of the probe. Immunohistochemical detection was accomplished with antidigoxigenin sheep F_(ab) fragments coupled to fluorescein (Roche Molecular Biochemicals), diluted 1:200 in 0.1 M Tris-HCL (pH 7.5), 0.15 M NaCl. The slides were washed in PBS, air dried, and stained with propidium iodide containing antifade (1,4-diazabicyclo[2.2.2]octane; Sigma, Munich, Germany). Fluorescence microscopy was performed with a Zeiss photomicroscope equipped with fluorescein epifluorescence filters. A tumor was considered to be MYCN-amplified if more than three nuclear signals were seen or if tumor nuclei showed relatively large signals, compared with accompanying fibroblasts or lymphocytes, which usually showed a pair of distinct nuclear signals. To adjust for potential hyperploidy, samples were cohybridized with a chromosome 2 centromeric probe (AppligeneOncor). The signals were considered to be specific if they could be consistently demonstrated in at least 50% of the tumor cells.

Statistics
All calculations were made using the SPSS software package, version 10.0 (SPSS Inc, Chicago, IL), run on a personal computer. ² statistics were used to analyze the associations between categorical variables. Categories of continuous variables were compared by means of the Mann-Whitney U test and the Kruskal-Wallis nonparametric analysis of variance.

The clinical outcome was analyzed as event-free survival (EFS) on the basis of any clinical evidence of relapse or disease-progression and as disease-specific survival (DSS) on the basis of all deaths in the presence of extensive tumor load after exclusion of causes of death unrelated to disease. Univariate survival analysis was computed by means of the Kaplan-Meier method, and significance levels were assessed by means of the log-rank test with statistical significance assumed at P < .05. The cutoff point for RIs was chosen at 10%, as proposed in an earlier investigation,³⁹ and patient age was dichotomized with a cutoff point at 12 months according to the established risk stratification for neuroblastomas. The significance of patient age and RI was verified by testing these factors as continuous variables in a univariate Cox regression analysis. All parameters shown by univariate analysis to be significant were also compared by means of a multivariate Cox regression analysis for multiple proportional hazards using a stepwise (backward as well as forward) conditional approach. Hughes’ grade was integrated as a multilevel factor, whereas INSS stage was dichotomized (stage 4 v all other stages) because there was no significant difference between the outcomes of stages 1, 2, 3, and 4S. The selection of independent factors was achieved with use of the maximum likelihood ratio. The multivariate analysis was conducted on the entire series (161 patients, without INPC grade as a covariate), on the 132 patients graded according to the INPC, and on the 29 patients who had received cytotoxic therapy before the diagnostic biopsy. For internal validation of the data, we used the so-called jackknife technique, which consists of successive reiterations of the multivariate analyses after exclusion of randomly chosen single patient cases (eg, patient cases 10, 25, 40, and so on, or patient case 8, 20, 32, and so on). Twenty-five models were built in this fashion. All P values are two-sided.

Because we previously identified the Ki-67 index as an independent prognostic indicator for neuroblastoma,¹⁴ we also verified the prognostic strength of the RI by repeating the analyses with inclusion of the Ki-67 index as a covariate in the multivariate models.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
A total of 161 neuroblastomas of all stages were analyzed in a blind-trial fashion by immunostaining for the repp86 protein using the monoclonal antibody Ki-S2. Immunolabeling for repp86 was consistently nuclear. Positive cells were often arranged in small clusters, but a diffuse distribution was also found. In the whole series, RIs ranged from 0% to 48% (median, 8%; mean, 10.2% ± 9.9%).

Correlation of RIs With Clinical, Histopathologic, and Molecular Parameters
RI values in relation to different groups defined by clinical, histopathologic, and molecular characteristics are surveyed in Table 1. There was no correlation between RI and the patients’ age, in contrast to a statistically significant correlation with INSS stage. On average, median RIs were low in stages 1 to 3, intermediate in stage 4S, and high in stage 4.

Like repp86, the MYCN status was assessed in a simple blind fashion with respect to clinical data. Analysis of the MYCN gene revealed 38 samples (23.6%) with amplification and 123 patients without apparent changes in copy number. Six to 9 copies of the MYCN gene were found in nine tumors, whereas 29 tumors contained 10 copies or more. The tumors with MYCN amplification exhibited a significantly higher median RI than did nonamplified tumors.

From a previous investigation¹⁴ we retrieved data on Ki-67 expression. The correlation coefficient between Ki-67 and repp86 labeling indices was r = 0.74 (P < .0001), indicating a close although by no means complete correlation.

Notwithstanding the statistical significance found for most of these correlations, a broad range of RI values was encountered in almost all of the above-defined categories and the RI value ranges were not strikingly different (Table 1). These data indicate that the different grades and stages of neuroblastoma are heterogeneous with respect to cellular proliferation.

Survival Analysis
Recurrence or tumor progression occurred in 68 patients (42.2%), and 57 patients (35.4%) died of the disease. Two patients rapidly succumbed to the chemotherapy without signs of tumor relapse or progression and were considered as censored data. Pretreated patients had a slightly poorer outcome (EFS, 0.49 ± 0.08; DSS, 0.52 ± 0.08) than those who were treated after diagnostic biopsy (EFS, 0.53 ± 0.05; DSS, 0.64 ± 0.05), but the differences were not statistically significant (P = .76 and P = .29, respectively).

In the univariate analysis, the established prognostic factors of patient age (P = .035 for EFS; P = .013 for DSS), INSS stage (P = .002 for EFS, Fig 1A; P = .0004 for DSS), Hughes’ histopathologic grade (P = .007 for EFS; P = .008 for DSS), INPC grade (P = .0004 for EFS, Fig 1B; P = .007 for DSS), and MYCN status (P = .0011 for EFS; P = .0005 for DSS) were significantly predictive of the clinical outcome. Patient age remained significant when examined as a continuous variable by univariate regression analysis (P = .025 for EFS; P = .017 for DSS). Compared with other grades according to Hughes’ classification, 1b tumors had the poorest prognosis, and the outcome for patients in stage 4 was markedly worse than for those in other stages (stage 4 v all other stages, P < .0001).

View larger version (18K): [in this window] [in a new window]   Fig 1. Univariate Kaplan-Meier analysis of event-free survival with stratification on International Neuroblastoma Staging System stage (A; 161 patients, P = .0018) and International Neuroblastoma Pathology Classification grade (B; 132 patients, P = .0004).

View larger version (16K): [in this window] [in a new window]   Fig 2. Univariate Kaplan-Meier analysis of event-free survival with stratification on the repp86 index in the entire cohort (A; 161 patients, P < .0001) and in the patients without pretreatment (B; 132 patients, P < .0001).

Neither the INPC criteria nor the MYCN status were as predictive of survival as the RI in different tumor stages or grades (data not shown). The prognostic effect of the RI also remained verified when risk groups were formed by combination of several standard prognostic factors. For example, in stage 4S tumors without MYCN amplification, events were exclusively observed in the group with a high RI, accounting for 62.5% of relapses and 50% of disease-related deaths (P < .0001). Conversely, for example, in stage 4 tumors with MYCN amplification, only one out of four patients with a low RI relapsed and eventually died of the disease, whereas in the group with a high RI, 16 of 17 patients relapsed, and 15 died of the disease (P < .0001).

To identify the independent prognostic factors for EFS and DSS, we performed a multivariate analysis using three different models. The first model covered the entire series of 161 cases and comprised patient age, INSS stage, Hughes’ grade, MYCN status, and the RI as covariates. In the second model, the INPC grade was included as an additional cofactor, which reduced the cohort to 132 cases. A third model was formed with the group of pretreated patients and logically excluded the INPC grade as a covariate. In the first two models, the RI emerged as the most influential predictor of both EFS and DSS, followed by INSS stage (Table 3). None of the other covariates were selected in this analysis. In the group of pretreated patients, INSS stage failed to achieve statistical significance, so that the RI remained the only independent predictor of EFS and DSS. Identical results were obtained with the backward and forward stepwise approach. In addition, the selection of independent prognostic factors was unchanged when the RI was examined as continuous (discrete) values instead of the categorical variable.

When the RI was excluded from the covariate list, only INSS stage and the MYCN status remained in the final selection. The odds ratios for disease recurrence or progression (EFS) were 4.09 (P < .0001; INSS stage 4) and 1.85 (P = .023; MYCN) in the entire cohort and 4.25 (P < .0001; stage 4) and 2.23 (P = .014; MYCN) in the series of 132 patients graded according to the INPC system. For the occurrence of tumor-related death, stage 4 was associated with a relative risk of 6.35 (P < .0001) in the entire series and of 6.63 (P < .0001) in the series of 132 untreated patients. MYCN amplification portended a relative risk for tumor mortality of 2.08 (P = .015) in the entire series and 2.35 (P = .022) after inclusion of INPC grade.

An internal validation was obtained using a jackknife technique for model building. Cox regression analysis selected the same independent factors in the subsets as formed, and the maximum divergence observed in the relative risk was 0.19 for a RI greater than 10%.

As an additional verification, we included the Ki-67 data available from a previous investigation¹⁴ as a covariate in the multivariate analysis. Although the results for the RI and INSS stage remained virtually unchanged (the maximum difference in the relative risk values for RI and INSS stage 4 was 0.11), a Ki-67 index more than 25% was selected as an independent prognostic factor in the series of 161 patients with relative risks of 2.27 (P = .0051) and 2.06 (P = .0062) for tumor progression and disease-related mortality, respectively. The Ki-67 index nevertheless lost its significance after inclusion of the INPC grade. The data are not presented in detail because they are outside the scope of this study.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Neuroblastoma is distinct from most malignant tumors because of its propensity for differentiation. Whereas the evolution of cancer in adults is generally accompanied by a gain of malignant qualities, a subset of neuroblastomas may mature to less aggressive phenotypes and may even undergo extensive regression. This indicates that neuroblastoma is a heterogeneous disease comprising a fraction of tumors that retain the properties of embryonal tissues with at least partly preserved mechanisms of growth regulation.^19,22

Differentiation typically goes along with a downregulation of cellular proliferation.⁴⁰ Accordingly, the proliferative activity, at least in terms of the mitosis-karyorrhexis index, has become an integrative constituent of several grading systems for neuroblastoma.^3,6,41,42 Moreover, the unmitigated tumor growth fraction, as assessed by means of the standard proliferation marker Ki-67,^43,44 was identified as an independent prognostic factor.^14,45 The Ki-67 index showed a closer correlation to the clinical outcome than patient age, tumor grade, and MYCN status but was only second in the hierarchy of independent factors (the first was tumor stage).¹⁴

In this study, we used an entirely different type of proliferation marker, repp86, which exclusively recognizes proliferating cells in the cell cycle phases S, G₂, and M.^24,28 We reasoned that this marker might more accurately reflect the biologically relevant fraction of proliferating cells because the fate of G₁ phase cells is uncertain.⁴⁰ These G₁ phase cells may indeed resume cycling but may remain in G₁ for an indeterminate period of time,⁴⁶ or they may definitively leave the cell cycle to become quiescent or succumb by apoptosis.⁴⁰ Once cells have crossed the restriction point and become engaged in DNA replication, however, they are likely to complete a division cycle because cell cycle progression depends on an intrinsic program that is largely refractory to external influences.⁴⁰

Consistent with our hypothesis, the RI emerged as the most powerful independent predictor of prognosis, superseding even tumor stage. It also seems that the prognostic effect of the RI is not significantly affected by cytotoxic treatment before the diagnostic biopsy, which could signify an additional advantage because one important prognostic criterion, the INPC grade, is not applicable to pretreated tumors. Moreover, the RI allowed discrimination between prognostically favorable and unfavorable patients in different age groups, all tumor stages and grades, and tumors with and without MYCN amplification. Even in stage 4S, which is known to have a favorable prognosis despite a heavy tumor burden,⁴⁷ an RI more than 10% characterized a pernicious phenotype, whereas none of the tumors with a lower RI recurred or progressed. This result is well in line with the alleged biologic heterogeneity of neuroblastoma,^1,19 for which apparently staging or grading systems, or even amplification of the MYCN gene, do not provide an accurate gauge.

In cancers of adulthood, it is rare to find a single factor that outweighs the prognostic effect of stage, which is the result of intrinsic properties of the tumor combined with the effects of a time course. In one earlier study, the Ki-S2 (repp86) index topped the hierarchy of prognostic factors for breast carcinomas,³⁹ but because only node-negative cases were examined, information on the effect of stage was limited. However, in endometrioid adenocarcinomas of the uterus, the RI was selected as the leading prognostic factor with a higher odds ratio than tumor stage.⁴⁸ This exceptional predictive strength, which was reproducible for neuroblastoma, is likely attributable to an association of the repp86-positive cell fraction with defects in cell cycle regulators and the expression of oncogenes²⁸ (Rudolph P, Olsson H, Borg A, et al, manuscript in preparation). Indeed, recent evidence indicates that repp86 plays a pivotal role in cell cycle promotion and mitotic cell division (Steffen R, Parwaresch R, Rudolph P, et al, submitted for publication). In this way, the RI might reflect the sum of genetic aberrations and biochemical dysfunctions leading to unbridled tumor growth.

Two other factors were reported to predict the prognosis of neuroblastoma with greater accuracy than tumor stage: gain of chromosome arm 17q¹⁵ and TA.¹⁷ In addition to a frequent association with MYCN amplification and deletions of a putative tumor suppressor gene on chromosome 1p32–36, the former apparently results in amplification and, consequently, augmented expression of yet unidentified tumor-promoting genes.¹⁵ Because such oncogenes often interfere with cell cycle regulation,^40,49 this idea further supports our interpretation of the biologic significance of repp86 expression.

Conversely, the tremendous prognostic effect of TA¹⁷ merits attention with a view toward understanding our results. Telomerase is an enzyme involved in cell immortalization that is downregulated in differentiating tissues while exhibiting substantial activity levels in most human cancers (see⁵⁰ for a recent review). Lack of TA might therefore reliably identify differentiating neuroblastomas, whereas sustained activity would indicate a propensity for tumor progression.²² TA is also invariably associated with cellular proliferation.²³ Interestingly, we observed that despite a fair correlation with the Ki-67 index, TA was much more closely associated with repp86 expression in different tumor types.^30,31 Notably, in a series of 38 neuroblastomas analyzed with the telomere repeat amplification protocol assay,²² the presence of TA was associated with an RI greater than 10% (P < .0001, Fisher’s exact test; unpublished results). The relative percentage of repp86-expressing tumor cells also predicted recurrence of endometrial carcinoma with a precision that was comparable, albeit not equivalent, to that of telomerase measurements.³¹ Although the underlying molecular mechanism could not yet be elucidated, the apparently momentous link between repp86 expression and TA might provide an additional explanation for the high prognostic significance of repp86 in neuroblastoma.

In conclusion, our study characterizes repp86 as a powerful prognostic factor in neuroblastoma. Assessment of repp86 expression may be of great assistance in resolving the biologic heterogeneity of neuroblastomas that seems to exist in all tumor stages and grades, in patients at any age, and in tumors with and without MYCN amplification. Our data indicate that repp86 expression may enable the identification of tumors that defy established prognostic models and may predict the tumor’s biologic behavior with great accuracy. If our findings are corroborated by other studies, immunostaining for repp86 might considerably refine the prognostic criteria for neuroblastoma. Because of the technical simplicity of the detection method and its perfect suitability for archival material, repp86 immunohistochemistry might advantageously replace telomerase measurements when fresh tumor material is unavailable for analysis.

	NOTES

 
Supported by grants from the Kinder Krebs Initiative, Buchholz Holm-Seppenesen, and the Else Kröner-Fresenius Stiftung, Bad Homburg, Germany.

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Submitted May 13, 2002; accepted January 20, 2003.

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