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Comprehensive Genetic and Histopathologic Study Reveals Three Types of Neuroblastoma Tumors

By Maria astowska, Catherine Cullinane, Sadick Variend, Simon Cotterill, Nick Bown, Seamus O’Neill, Katia Mazzocco, Paul Roberts, James Nicholson, Caroline Ellershaw, Andrew D.J. Pearson, Michael S. Jackson, for the United Kingdom Children Cancer Study Group and the United Kingdom Cancer Cytogenetics Group

From the Human Genetics Unit, School of Biochemistry and Genetics; and Institute of Child Health, University of Newcastle upon Tyne, Newcastle upon Tyne; Department of Histopathology; and Cytogenetics Unit, St James’s Hospital, Leeds; Department of Histopathology, Sheffield Children’s Hospital, Sheffield; Wessex Regional Laboratory, Salisbury; United Kingdom Children Cancer Study Group, Department of Epidemiology and Public Health, University of Leicester, Leicester; Department of Paediatrics, Addenbrooke’s Hospital, Cambridge, United Kingdom; and Unit of Solid Tumour Biology, Advanced Biotechnology Center, Genova, Italy.

Address reprint requests to Maria astowska, Human Genetics Unit, School of Biochemistry and Genetics, University of Newcastle upon Tyne, Ridley Building, Claremont Place, Newcastle upon Tyne NE1 7RU, United Kingdom; email: M.A.Lastowska{at}ncl.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine the relationship between multiple genetic features, tumor morphology, and prognosis in neuroblastoma.

PATIENTS AND METHODS: The genetic alterations and morphologic features that underpin three histopathologic risk classifications were analyzed in 108 neuroblastoma patients. Tumors were subdivided into four groups based on the three most frequent and prognostically significant genetic alterations (17q gain, 1p deletion, and MYCN amplification), and all other genetic, morphologic, and clinical data were analyzed with respect to these groups.

RESULTS: Our analyses identify three nonoverlapping tumor types with distinct genetic and morphologic features, defined here as types 1, 2, and 3. Type 1 tumors show none of the three significant genetic alterations and have good prognosis. Both type 2 (17q gain only or 17q gain and 1p del) and type 3 (17q gain, 1p del, and MYCN amplification) tumors progress. However, these tumor types are distinguished clinically by having significantly different median age at diagnosis and median progression-free survival (PFS). Multivariate analysis indicates that 17q gain is the only independent prognostic factor among all genetic, histopathologic, and clinical factors analyzed. Among histopathologic risk systems, the International Neuroblastoma Pathology Classification was the best predictor of PFS.

CONCLUSION: Our results indicate that specific combinations of genetic changes in neuroblastoma tumors contribute to distinct morphologic and clinical features. Furthermore, the identification of two genetically and morphologically distinct types of progressing tumors suggests that possibilities for different therapeutic regimens should be investigated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
NEUROBLASTOMAS, THE most common extracranial solid tumors of childhood, are characterized by diverse behavior ranging from rapid malignant progression to spontaneous regression. As a result, both prognosis and response to therapy can vary widely.^1,2 Extensive efforts have been made to use clinical criteria to select appropriate therapy and have led to the proposal for standardization of criteria for diagnosis, staging, and response to treatment known as the International Neuroblastoma Staging System (INSS).³ However, it is clear that clinical criteria are not always sufficient to predict disease outcome, and the current international recommendation is that data regarding biologic features should be collected on all patients so that new therapeutic groups can be defined.³

Genetic abnormalities that characterize this disease have been known for some time,^4-8 and several are of clear prognostic value. Indicators of poor prognosis include diploidy or tetraploidy,^9,10 MYCN amplification,^11-13 deletion of 1p,^14-17 and gain of chromosome arm 17q.^18-21 The latter rearrangement, which can result from unbalanced translocation of 17q to more than 20 different chromosome regions,^22,23 is the most powerful genetic predictor of adverse outcome in neuroblastoma.^24-26 In addition, frequent loss of heterozygosity (LOH) has been reported for other chromosomal regions in neuroblastoma, specifically 2q (30%), 3p (15.3%), 4p (19.5%), 9p (36%), 11q (5% to 44%), 14q (18% to 23%), and 18q (31%).^27-33 Among these, losses at 3p, 9p, and 11q may also be prognostically valuable.^29,31,32

To date, strong associations have been identified between MYCN amplification and 1p deletion,^34-36 and between 17q gain, 1p deletion, and MYCN amplification.^24,26,37,38 In contrast, losses at 11q are inversely correlated with MYCN amplification,³² and losses at 14q are inversely correlated with both MYCN amplification and 1p deletion.^27,33 Furthermore, comparative genetic hybridization (CGH) studies have revealed an association between loss of 11q and 3p³⁹ and identified a subset of advanced tumors with 11q, 14q, and 3p losses but with no 1p deletion or MYCN amplification.²¹

In addition to genetic features, a valuable insight into tumor biology has been obtained using histopathologic analyses.^40-43 In 1984, Shimada et al⁴⁴ introduced a classification system based on the amount of schwannian stroma, degree of differentiation, mitotic-karyorrhectic index (MKI), and age at diagnosis. This formed the basis of a new prognostic categorization which divided tumors into favorable (FH) and unfavorable histopathology (UH). In 1992, Joshi et al^45,46 introduced risk groups based on three histologic grades and patient age that have subsequently been refined as modified grades and, in consequence, are referred to as Modified Risk Groups.⁴⁷ Finally, a new International Neuroblastoma Pathology Classification (INPC), based on the original Shimada classification with minor modifications, has been recommended.^48,49

With the existence of both genetic prognostic factors that show nonrandom distribution patterns and histopathologic prognostic indicators, it is clear that integrated analyses of multiple features represents a logical and potentially valuable approach to understand disease progression and to identify subgroups as targets for specific therapeutic regiments. However, the few studies that have linked genetic factors with histopathology have analyzed MYCN,^12,49,50 and more recently 11q and 1p,^32,36 without reference to other genetic factors. In this study, we characterize both genetic abnormalities and the morphologic features that underpin three histopathologic classifications (original Shimada, INPC, and Modified Risk Groups) in 108 primary tumors to allow the relative importance of all well-established prognostic features to be evaluated within the same group of patients. This has also allowed us to investigate and identify associations between genetic and morphologic features of potential biologic significance.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
Patients presented between October 1987 and December 1999 at any one of 11 United Kingdom and Republic of Ireland centers, with patient age ranging from 1 month to 18 years. More than 80% of cases were diagnosed within the last 5 years of the study, from 1995 to 1999. Neuroblastoma diagnosis and staging was according to INSS,³ the distribution of stages being as follows: stage 1 (11 patients), 2 (10 patients), 3 (18 patients), 4 (61 patients), and 4s (eight patients). Primary tumors from 108 patients were studied before therapy. Therapy was administered according to current protocols (the United Kingdom Children’s Cancer Study Group [UKCCSG], European Neuroblastoma Study Group, and Localized Neuroblastoma European Study Group) and similar treatment was administered in all contributing centers. The sole criterion for inclusion in the series was technical success in establishing chromosome 17q, MYCN, and 1p status within tumor material from the same site as the sample that underwent histopathologic assessment. In eight of 108 cases, information about MYCN and/or 1p only was available, but these tumors have been included to allow correlation between MYCN status with CD44 expression.

Histopathology
The histologic diagnosis, based on recent INPC recommendations,⁴⁸ was reviewed centrally by two UKCCSG pathologists (C.C. and S.V.) with no knowledge of the genetic abnormalities of each tumor. Tumor material from all patients diagnosed before 1999 was reanalyzed to ensure consistency with INPC recommendations. The following morphologic features were investigated: amount of schwannian stroma, MKI (low, medium, and high being < 100, 100 to 200, and > 200 mitotic or karyorrhectic cells per 5,000 tumor cells, respectively), degree of differentiation based on presence or absence of neuropil, ganglionic differentiation (undifferentiated, poorly differentiated, or differentiating), and the presence or absence of calcification. Results of CD44 expression were already available for 35 patients and were detected immunohistochemically using the CD44 antibody (DAKO). Because the expression of this cell surface glycoprotein reflects tumor phenotype and is a favorable prognostic indicator,⁵¹ we have included this information. The distribution of single morphologic features within the sample, and the resulting histopathologic risk groups, is listed in Table 1.

Statistical Analysis
Comparison of median age at diagnosis and median numerical and structural chromosome imbalances were made using the rank sum (Mann-Whitney) two-sample test. Fisher’s exact Test (two-sided) was used for comparing variables with two levels and the ² test for trend was used for comparing proportions with three or more levels. No adjustments were made for multiple testing. Survival and progression-free survival (PFS) were calculated using Kaplan-Meier estimation,⁵⁶ and group comparisons were made using the log-rank test, except for comparisons between groups 2 to 4, which were made using the Wilcoxon (Breslow) test. Backward step-wise Cox proportional hazards procedures were used to determine which variables had an independent effect on PFS. In addition, major genetic features (17q gain, 1p deletion, and MYCN amplification) were tested to see if they provided any additive prognostic value when modeled with each of the histopathology classification systems.

	RESULTS

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Genetic Characteristics of Tumors
Gain of chromosome arm 17q, MYCN amplification, and 1p deletion are the only structural rearrangements of proven prognostic significance and high frequency in neuroblastoma. We therefore divided the tumor series into genetic groups based on this information (Fig 1). If these three genetic alterations were assorted randomly, it would be possible to identify eight genetically distinct subgroups. However, 92 out of the 96 tumors that were unambiguously informative for all three genetic alterations fell into the following four groups: group 1, structurally normal chromosome 17, no 1p deletion, and no MYCN amplification (29 patients); group 2, 17q gain, no 1p deletion, and no MYCN amplification (24 patients); group 3, 17q gain, 1p deletion, and no MYCN amplification (12 patients); and group 4, 17q gain, 1p deletion, and MYCN amplification (27 patients). The four remaining tumors had the following combinations of abnormalities: no 17q gain, 1p deletion, and no MYCN amplification (one tumor); no 17q gain, 1p deletion, and MYCN amplification (two tumors); and 17q gain, no 1p deletion, and MYCN amplification (one tumor). These were not included for further subdivisions and analysis. Because approximately 95% of all tumors in our series fell within one of four groups defined by the three well-established genetic prognostic features, we analyzed all other genetic, morphologic, and clinical features with respect to these subgroups.

View larger version (17K): [in this window] [in a new window]   Fig 1. Relationship between 17q, 1 p and MYCN in 96 neuroblastoma tumors. Four tumors with mixed clones (both unbalanced 17q gain and whole chromosome 17 gain) were excluded from presentation.

View larger version (18K): [in this window] [in a new window]   Fig 2. Overall survival (A) and PFS (B) for neuroblastoma patients from genetic groups 1 to 4. Median PFS for group 2: 28 months; for group 3: 14 months; for group 4: 9 months. Wilcoxon (Breslow) test: group 2 v group 3, P = .2; group 2 v group 4, P = .04; group 3 v group 4: P = .7.

To specifically evaluate the histopathologic risk systems in our series of patients, all three were included in a backwards step-wise Cox regression analysis of PFS. The INPC system is significant (hazards ratio, 14.2; P < .001), whereas the original Shimada and Modified Risk Group systems can be rejected (P > .05). The power of the three principal genetic abnormalities relative to the three histopathologic risk systems was also tested, and these results are listed in Table 4. Gain of 17q adds prognostic value to all three schemes, 1p deletion adds value to the Shimada original system but not the INCP or Modified Risk Group systems, and MYCN does not add prognostic value to any of the classification systems.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have classified 96 tumors in terms of three genetic prognostic indicators. Greater than 95% of the tumors fell into one of four genetically distinct groups. This distribution is similar to the results obtained in the analysis of 260 tumors described by Bown et al²⁶ and suggests that our series is representative. Analysis of further genetic and histopathologic features indicated that two of the four genetic groups (groups 2 and 3) could not be distinguished from each other statistically. As a result, we can distinguish a minimum of three nonoverlapping types of neuroblastoma from this analysis.

Type 1 tumors (genetic group 1; regressing) have mainly numerical changes and a triploid number of chromosomes. Such tumors have been distinguished previously as type 1 by Brodeur et al⁵⁷ and by Maris and Matthay.⁵⁸ In the present study, however, it was found that this group is also significantly associated with losses of whole chromosomes 4, 14, and 3, gains of chromosomes 17 and 7, low MKI, calcification (present in > 50% of cases), positive CD44 expression, absence of undifferentiated cells, and INPC favorable histopathology. Patients are mainly infants with low-stage disease and with excellent survival rate.

Type 2 (genetic groups 2 and 3; progressing) is distinguished from type 1 by having a large number of structural abnormalities, including frequent 11q deletion, INPC unfavorable histopathology, older age of patients, advanced stages of disease and poor prognosis.

Type 3 (genetic group 4; rapidly progressing) is characterized by an absence of 11q deletion, few other deletions, negative CD44 expression, and absence of calcification. In addition, only tumors of this type exhibited an undifferentiated morphology. Median age at diagnosis was lower (2.3 years) and median PFS shorter (9 months) than in type 2 tumors. Because this group is defined by the presence of MYCN amplification and there are no further genetic features specific to this group, MYCN amplification is the most obvious candidate for the observed alteration in tumor morphology.

Although there is clear statistical support for the three distinct tumor types defined above, there are also clinical trends within type 2 tumors that suggest that group 3 tumors may be more aggressive than group 2. Group 3 tumors had shorter median survival than group 2 (14 months v 28 months, respectively), no low-stage disease, and a younger age at diagnosis (1.8 years v 4.0 years, respectively). These differences could be due to the influence of 1p deletion (which defines group 3) and suggest that 1p deletion can influence tumor progression but not morphology. However, the main conclusion from these observations must be that further studies with larger sample sizes are needed to clarify the clinical difference between these two groups and allow the definition of a fourth tumor type based on the genetic stratification used here. It is possible that some results with borderline significance are due to multiple testing. However, most of the results have a high level of statistical significance despite the small sample size, and the use of two-sided tests for association have a conservative effect on the results. Moreover, the three tumor types we have identified are each distinguished by multiple significant differences.

To date, all analyses of the relationship between genetic and morphologic features have concentrated on single genetic abnormalities, usually MYCN amplification. Shimada et al⁴⁹ revealed that MYCN amplification was associated with UH, and this is confirmed in our study. Furthermore, in our series, all undifferentiated tumors had MYCN amplification. A correlation between MYCN amplification and lack of differentiation was noticed earlier using older criteria for tumor differentiation.^12,50 An association between MYCN amplification, high MKI, and a high percentage of cells in S phase has been previously reported.^50,59 Our group of patients with MYCN amplification is heterogeneous with regard to MKI, ranging from low to high. It is impossible to envisage which genetic change would influence MKI in these tumors, because all of them had 17q gain, 1p deletion, and very few other changes. It is possible, for instance, that genes coamplifying with MYCN (eg, DDXI, which coamplifies in up to 50% of cases)^60,61 may have an impact on proliferative potential. Finally, the association between MYCN amplification and negative CD44 expression identified here has been reported before.^62,63 Correlation between lp status and histopathology has focused on selected stroma-rich tumors,⁶⁴ although a very recent study of a large number of patients indicates that 1p LOH is associated with Shimada UH.³⁶ Unfavorable histopathology has also been associated with 11q LOH but only in single copy MYCN tumors.³² These results are in concordance with our findings that both type 2 and type 3 tumors are mainly of UH and are frequently deleted for 1p, whereas type 2 tumors are frequently deleted for 11q.

The presented above extensions to the known associations between genetic, histopathologic, and clinical features has allowed us to develop a model of neuroblastoma development, and this is presented in Fig 3. Group 1 tumors seem to follow a genetically distinct pathway that determines the ability of these tumors to respond to therapy. In contrast, groups 2 to 4 are the progressing tumors with heterogeneous characteristics. Because 17q gain is the only feature that underpins all three groups, it is possible that a critical genetic alteration for the acquisition of metastatic potential and/or invasiveness in neuroblastoma is partial gain of 17q. Group 4 tumors progress rapidly, are distinct morphologically from all other groups, and accumulate few other genetic abnormalities. By contrast, group 2 tumors seem to grow more slowly and accumulate secondary genetic abnormalities, presumably because of the time available. Because der(1)t(1;17)(p;q) was identified in nearly 50% of all analyzed unbalanced translocations of 17q in primary neuroblastoma tumors,⁶⁵ we present two pathways by which 17q is gained. Moreover, it is likely that deletion of 1p resulting from der(1)t(1;17)(p;q) involves a larger region in group 4 than in group 3 because a different pattern of LOH on 1p was associated with MYCN amplified and not amplified tumors. It would further imply that in both groups distinct tumor suppressor genes on 1p may be involved (tsg1? or tsg2?).^35,66-68

View larger version (66K): [in this window] [in a new window]   Fig 3. Neuroblastoma characteristics based on genetic, morphologic, and clinical features.

Our results may also have important clinical consequences. First, we found that only the most recent differentiation classification (within INPC system) was of prognostic value. It is, therefore, not surprising that we found the INPC system^48,49 to be more powerful than both the Modified Risk Groups and original Shimada classifications, which are both based on the old evaluation of differentiation. This is, to our knowledge, the first analysis to assess the value of this new system.

Second, notwithstanding the clinical importance of the INPC classification, multivariate analyses indicated that 17q gain is a more powerful prognostic indicator than the histopathologic risk systems we analyzed and confirms the previous finding that 17q gain is a more powerful prognostic indicator than all other genetic and clinical features analyzed.²⁶ This is the first time that the relative importance of 17q gain and morphologic features has been evaluated and further strengthens suggestions that 17q status should be routinely evaluated.^24,26

Third, we find that although negative CD44 expression identifies a subset of tumors with poor prognosis (type 3), positive CD44 expression is not helpful in the prediction of outcome because nearly all analyzed tumors from type 1 (prognostically favorable) and type 2 (prognostically unfavorable) have positive CD44 expression. Although only five type 1 tumors with CD44 results are presented here, the analysis of Favrot et al⁵¹ found that 100% of low-stage disease show positive CD44 expression.

Finally, the identification of at least two types of aggressive tumors may have important clinical implications. Type 2 is characterized by significantly older age at diagnosis, the presence of some low-stage tumors and longer median survival, caused by later relapses and deaths, compared with type 3 tumors. This is particularly true, and supported statistically, for tumors from genetic group 2 (Table 3). This would imply that tumors of this type, although retaining potential for metastasis and recurrence, may behave differently as a consequence of their distinct genetic profile. However, the majority of patients from type 2 and type 3 have stage 4 disease and are currently treated in the same way. It is reasonable to suggest that future therapeutic schemes could be investigated separately for both groups of patients.

	ACKNOWLEDGMENTS

 
Supported by the North of England Children’s Cancer Research Fund.

We thank the following UKCCG members for providing karyotype and/or FISH information: Liz Sinclair and Jill Elliot, Sheffield; Lindsay Paterson, Glasgow; Mike Griffith and Dominic McMullan, Birmingham; Willemijn van de Klundert and Rod Howell, Bristol; Kate Martin, Nottingham; Jane Fennel, Manchester; Andy Pearce, Edinburgh; Fiona Ross, Salisbury; and Mark McKinley, Anne Keen McGuire, and Eddy Mahler, Oxford, United Kingdom. CGH results from Dublin were provided by Raymond Stallings and have been included in a previous publication.³⁹ We also thank all of the surgeons, pathologists, and pediatric oncologists from the contributing UKCCSG centers.

	REFERENCES

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Submitted December 29, 2000; accepted April 3, 2001.

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