mycn protein expression as a predictor of neuroblastoma...

Vol. 3, 1699-1706, October 1997 Clinical Cancer Research 1699

3 The abbreviations used are: VMA, vanillymandelic acid; HVA. ho-

movanillic acid.

MYCN Protein Expression as a Predictor of Neuroblastoma

Prognosis1

Helen S. L. Chan,2 Brenda L. Gallie,

Gerrit DeBoer, George Haddad, Naohiko Ikegaki,

Jim Dimitroulakos, Herman Yeger, and

Victor Ling

Division of Hematology-Oncology and Immunology, Department of

Pediatrics [H. S. L. C., G. HI, Division of Immunology and Cancer

Research, Research Institute [H. S. L. C.. 0. H., B. L. G.], and

Departments of Ophthalmology and Molecular and Medical Genetics

[B. L. G.] and Pathology [J. D., H. Y.]. The Hospital for SickChildren and University of Toronto, Toronto, Ontario, M5G lX8

Canada; Division of Clinical Trials and Epidemiology, Toronto-

Sunnybrook Regional Cancer Center. Department of Medical

Biophysics, University of Toronto. Toronto, Ontario, M4N 3M5

Canada 1G. Dl: Departments of Pathology and Biochemistry, British

Columbia Cancer Agency, University of British Columbia,

Vancouver, British Columbia, V5Z lL3 Canada IV. LI: and

Department of Pediatrics, University of Pennsylvania School of

Medicine, Philadelphia. Pennsylvania 19104 IN. 1.1

ABSTRACT

About half of nonlocalized neuroblastomas have MYCN

gene amplification and usually progress rapidly, but the half

without such amplification also do poorly, albeit progressingmore slowly. We hypothesize that overexpression of MYCN

protein can occur without gene amplification and that this

expression reliably predicts the prognosis of neuroblastoma.

To determine whether MYCN expression correlated withoutcome, we assayed MYCN protein immunohistochemi-cally in 180 archival pretreatment and posttreatment sam-

ples and stratified the 57 conventionally treated stage IVS,

III, and IV patients by these conventional prognostic fac-tors: stage, age, serum ferritin, Shimada histology, urinary

catecholamine ratio, and MYCN gene status. At a median

Received 3/3/97; revised 6/18/97; accepted 7/2/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I Supported by grants from the National Cancer Institute of Canada with

funds provided by the Canadian Cancer Society (to H. S. L. C.. B. L. G..

V. L., and H. Y.), Medical Research Council of Canada (H. S. L. C.,

B. L. G.. and V. L.), USPHS Grant CA-37130 from the NIH (to V. L.),

Sandoz Canada, Inc., the Hospital for Sick Children Pediatric Consult-

ants and Research Institute, Atkinson Charitable Foundation, Elsa U.

Pardee Foundation (H. S. L. C.), and Canadian Genetic Diseases Net-

work, Retinoblastoma Family Association, and Royal Arch Masons of

Canada (B. L. G.). B. L. G. is a distinguished Scientist of the Medical

Research Council of Canada. This work was performed while

H. S. L. C. was a Research Scientist of the National Cancer Institute of

Canada supported by funds from the Canadian Cancer Society.

2 To whom requests for reprints should be addressed, at Division of

Hematology-Oncology, The Hospital for Sick Children. 555 University

Avenue. Toronto, Ontario, M5G lX8 Canada. Phone: (416) 813-5872;

Fax: (416) 8 13-5327; E-mail: [email protected].

follow-up of �6.8 years, we found in patients with known

MYCN gene status that the 23 of 37 without gene amplifica-

tion fared no better than the 14 of 37 with gene amplification

(P = 0.35 and 0.21, comparing relapse-free and survival

rates). Conversely, in patients without MYCN gene amplifi-

cation, 9 of 23 were found to overexpress MYCN protein

pretreatment, and they did worse than the 14 of 23 without

detectable MYCN protein (P = 0.0016 and 0.022, comparingrelapse-free and survival rates). Furthermore, MYCN pro-

tein expression was prognostic without (P = 0.00001 ) and

with (P = 0.0007) stratifying all 57 patients by MYCN gene

status, each conventional prognostic factor (P ranging from

0.00001-0.013), or simultaneously by the two most impor-

tant factors, stage and age (P = 0.00076). We conclude that

overexpression of MYCN protein without gene amplification

correlated significantly with the clinical behavior of neuro-

blastoma and predicted outcome independently of other

prognostic factors. This strongly supports the hypothesis

that expression of the MYCN oncogene is critical for pro-

gression of neuroblastoma.

INTRODUCTION

Neuroblastoma is a major cause of pediatric cancer mor-

tality. Stage and age broadly define prognosis in patients that

require different therapy. Patients of any age with localized

stages I and II generally do well and are curable by surgery.

Young infants with disseminated stage IVS are often cured with

minimal or no therapy, but older children with nonlocalized

stages III and IV do poorly, with a 13-38% cure rate despite

intensive therapy ± bone marrow transplant ( I , 2).

Age is an important conventional prognostic factor (3).

Stage and serum ferritin reflect the tumor load (3), and histol-

ogy, urinary catecholamines VMA3:HVA ratio, serum neuron-

specific enolase, ganglioside GD2, and irk oncogene expression

define the degree of tumor differentiation (3-6). Ploidy; muta-

tions of chromosomes lp, I I, 14, or 17 and neuropeptide Y:

bcl-2 apoptosis-suppressing protein; or nm23 metastasis-related

protein expression may reflect the aggressive potential (7-12).

We and others have observed that P-glycoprotein expression

confers poor prognosis to neuroblastoma (13-16), probably by

promoting broad-spectrum efflux of important cytotoxins from

tumor cells. Other investigators have reported a similar finding

for the putative multidrug resistance protein MRP (16, 17).

Amplification of the MYCN oncogene causing MYCN pro-

tein overexpression occurs in about half of the nonlocalized

neuroblastoma (12, 18). Such tumors usually relapse quickly,

suggesting that the MYCN gene plays an important role in the

progression ofneuroblastoma (12, 18, 19). However, the half of

Research. on July 14, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1700 MYCN Protein Predicts Neuroblastoma Prognosis

the nonlocalized tumors without such amplification also do

poorly in the long term (15-17). Because we hypothesize that

overexpression of MYCN protein occurring without MYCN

gene amplification might affect prognosis, possibly through the

activation of transcription of genes causing tumor progression,

we asked whether MYCN protein per se relevantly predicts the

clinical behavior of neuroblastoma.

PATIENTS AND METHODS

Patient Selection. We included 80 patients, ages 0.1-

13. 1 years, from the Hospital for Sick Children in Toronto

(1964-1990), in whom � I pretreatment samples were available

for MYCN protein evaluation. We staged the 23 localized

(stages I and II) and 57 nonlocalized tumors (27 stage IV, 21

stage III, and 9 stage IVS) according to the Evans classification

(3) by reviewing the records, radiographs, bone scan, comput-

erized tomography, bone marrow aspirates, and tumor biopsies.

We assessed conventional risk factors including stage, age,

serum ferritin, urinary VMA:HVA ratio, and tumor histology

according to the Shimada classification and determined the

MYCN gene status in the 37 stage IVS, III, and IV cases (47

samples) in which there was enough tumor for Southern blotting

(18). However, analysis of ploidy, chromosome lp, or newer

putative prognostic factors was unavailable in this retrospective

study. We tested the 23 stage I and II tumors (35 pretreatment

samples) in comparison with the 57 stage III, IV, and IVS

tumors (89 of 187 pretreatment samples, 98 of 187 during

therapy; mean, 3 samples/patient; range, 1-9). We grouped

stage IVS with stages III and IV rather than stages I and II,

because some IVS tumors without MYCN gene amplification

also expressed MYCN protein, and these infants progressed

despite intensive therapy, whereas none with stage I/Il tumors

progressed. Thus, stage IVS is probably a heterogeneous group

consisting of misdiagnosed IV as well as true IVS. The IVS

numbers are also so small that including or excluding them has

a minimal effect on the statistical analysis. We excluded patients

with ganglioneuroma, palliative cases, and consultations or re-

ferrals for bone marrow transplant only. We also excluded 35

localized and 76 nonlocalized tumors (3 1 stage IV, 28 stage III,

and 17 stage IVS) particularly from the earlier era, in whom

pretreatment samples were unavailable for testing (biopsies

were too small or were not retrievable from referral hospitals, or

no biopsies had been performed because the radiology, urinary

catecholamines, and/or bone marrow were considered to be

diagnostic for neuroblastoma).

Treatment. All patients were treated with surgery ±

radiation according to their stage and with the conventional

chemotherapy of their era: vincristine with cyclophosphamide

or teniposide (stage IVS), vincristine and cyclophosphamide

with dacarbazine or doxorubicin (stage III); and cyclophospha-

mide, doxorubicin, cisplatin, and teniposide ± bone marrow

transplant (stage IV; Refs. I and 2). The response to chemo-

therapy was judged as favorable if a complete (100%) or very

good partial response (�90%) was observed and as unfavorable

if a partial response (�50%) was observed or if there was

disease progression, and relapse-free and survival rates were

assessed from diagnosis to relapse, death, or last follow-up. On

average, living patients were followed through April 1995.

MYCN Protein Assay. Each sample was stained three

times by a sensitive multilayer immunoperoxidase assay similar

to the one we described previously for P-glycoprotein (15),

using both well-characterized monoclonal anti-MYCN antibod-

ies directed against separate NH, terminus epitopes (NCM II

100 and NCM IX 102; Ref. 20), with IgGl as the isotype-

matched negative control. Specificity was confirmed in selected

cases by blocking antibody-binding with the MYCNIMYCC

fusion protein from which the antibodies were raised (20). This

assay is not quantitative, although we have standardized the

staining against the MYCN protein content quantified by im-

munoblot (21) in negative and positive controls (SK-N-SH line

without MYCN gene amplification, and GOTO, NUB6, NB 1,

NUB32, IMR-32, and NUB7 lines with 39-380 copies of the

MYCN gene). SK-N-SH, in which MYCN protein was undetect-

able by immunoblot, was scored as negative, whereas GOTO,

NUB6, NB1, NUB32, IMR-32, and NUB7, with 18-100%

MYCN protein relative to NUB7, arbitrarily designated as

100%, were scored semiquantitatively as 1 + to S + for MYCN

protein staining (21-23). Three observers masked to sample

identity scored samples containing no MYCN-expressing tumor

cells as negative and designated those with any MYCN-express-

ing cells as 1 + to 5+ by their highest positivity in individual

cells, compared against the negative and positive controls. The

final score of each sample was based on the consensus of

observers or on the majority score, if the interpretation was not

unanimous. Only patients without any positive samples were

called MYCN protein negative, whereas those with � 1 positive

samples were designated MYCN protein positive.

Statistical Analysis. Response rates were compared by

Fisher’s exact test, and actuarial relapse-free and survival rates

were compared by the Kaplan-Meier method (24). To avoid

possible bias by confounding risk factors, each conventional

prognostic factor was inspected for uniformity of distribution in

the nonlocalized MYCN protein-positive and -negative groups,

and these patients were stratified according to each parameter.

Whether each parameter significantly affected outcome was

determined by log-rank analysis, without and with stratification

for the effect of MYCN protein (25). The effect of MYCN

protein was similarly assessed directly by stratifying for each

prognostic factor and simultaneously by stage and age. To avoid

possible selection bias, we evaluated all 133 nonlocalized cases

from 1964-1990, comparing survival rates within each stage for

study versus nonstudy patients in whom pretreatment samples

were not retrievable for testing. All statistical tests used were

two-sided.

RESULTS

The pattern and levels of MYCN protein showed consid-

erable heterogeneity between different samples that were ob-

tamed from different sites, at the same or different time points

in individual patients, and between different patients (Fig. 1).

We graded samples as I + to 4+ for staining of MYCN protein

in the nuclei of tumor cells, but we found no 5 + samples. In the

35 samples from the 23 localized cases, all of which had

favorable outcome, we found that MYCN protein was rarely

expressed (0 of 2 for stage I, and 1 of 21 for stage II). The

MYCN gene was confirmed to be nonamplified in 14 of these



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� ‘ �A � � �BL�Fig. 1 Multilayer immunoperoxidase staining of the MYCN nucleoprotein in neuroblastoma (hematoxylin counterstain, X I 200). The pattern andlevels of MYCN protein were heterogeneous. A, absence of detectable MYCN protein in a tumor without MYCN gene amplification; B, MYCN proteinlevel that was scored as 4+ in a tumor with 300 copies of the MYCN gene; C, MYCN protein level that was scored as 3 + in a tumor without MYCN

gene amplification.

localized tumors, including the stage II tumor in which MYCN

protein was detectable. In contrast, in the 89 pretreatment sam-

ples from the 57 nonlocalized cases, we found that the MYCN

protein was frequently overexpressed, more so in stages IV (IS

of 27) or III (6 of 21) than in stage IVS (0 of 9). There seemed

to be a higher incidence of MYCN protein expression during

therapy, particularly in stages IV ( 17 of 27) or III (9 of 21)

compared to stage IVS (1 of 9), when taking into account all 187

samples obtained throughout the course of disease. Initially

positive tumors continued to be positive at relapse, but some

tumors in which MYCN protein was undetectable before treat-

ment showed detectable MYCN protein in subsequent biopsies.

All IgG 1 controls were negative. The interobserver agreement in

interpreting whether MYCN expression was positive or negative

was unanimous for 91% of the patients and 69% of the samples.

We observed MYCN protein overexpression relatively fre-

quently, irrespective of whether the MYCN gene was amplified

in a particular tumor. Among the 37 nonlocalized cases in which

there was enough tumor for Southern blotting, we found MYCN

protein overexpression overall (59%; 14 of 20 for stage IV and

8 of 14 for stage III) more often than amplification of the MYCN

gene (38%; 8 of 20 for stage IV and 6 of 14 for stage III); both

occurrences were absent in the 3 stage IVS cases.

In contrast, undetectable MYCN protein expression was

relatively uncommon in the presence of gene amplification.

Among the 14 patients with MYCN gene amplification, we

detected MYCN protein before treatment in all but 3 tumors,

and all but 1 tumor subsequently. Two were stage III (25 and

200 copies of MYCN gene), in which MYCN protein was

undetectable at diagnosis in one sample of each but was ulti-

mately detectable at relapse in one and three samples, respec-

tively; both of these patients died of disease. The third was stage

IV (50 copies of the MYCN gene), in which MYCN protein was

undetectable at diagnosis in one sample; this tumor could not be

retested, because the 7.7-year survivor never relapsed.

Most importantly, MYCN protein overexpression could

still be observed relatively frequently, despite the absence of

MYCN gene amplification in a particular tumor. Among the 23

cases without MYCN gene amplification, we detected MYCN

protein before treatment in 6 tumors (5 of 12 stage IV, 1 of 8

stage III, and 0 of 3 stage IVS tumors) but in a total of 9 tumors

followed throughout the course of disease (7 of 12 stage IV, 2

of 8 stage III, and 0 of 3 stage IVS tumors).

We correlated MYCN protein expression with the din-

ical outcome of all 57 stage IVS, III, and IV patients and

found that it strongly predicts the long-term response to

therapy. At a median follow-up of 8.8 years, the 36 patients

with undetectable MYCN protein before treatment did better

than the 21 with overexpressed MYCN protein (67 versus

15% relapse-free, P = 0.00014; 64 versus 28% surviving,

P = 0.0047). Even when we excluded the nine better-prog-

nosis stage IVS patients, MYCN protein expression still

significantly predicted outcome in the 48 stage III and IV

patients (data not shown). We also observed a similar corre-

lation for patients with undetectable compared with increased

MYCN protein, taking into account all samples obtained

throughout the course of disease (Fig. 2, A and B). Any level

of MYCN protein expression apparently adversely affected

prognosis, because outcome was as poor with 1 + (7% re-

lapse-free; 13% surviving) as it was with 2+ (20% relapse-

free; 40% surviving), 3+ (40% relapse-free; 20% surviving),

or 4+ MYCN protein (0% relapse-free; 0% surviving). No-



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Fig. 2 Outcome of all 57 stage IV, III, and IVS neuroblastoma patients by MYCN protein expression, taking into account all samples obtainedthroughout the course of disease. Kaplan-Meier curves of relapse-free survival (A) and overall survival (B), showing a significant difference for 27patients with detectable MYCN protein compared with 30 patients with undetectable MYCN protein (P = 0.00001 and 0.00002; median follow-up,8.8 years). Two patients (one from each group) that died without relapse were treated as censored observations; this explains why the relapse-freesurvival was 80%, whereas the overall survival was 79%.

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Fig. 3 Kaplan-Meier curves of 37 stage IV, III, and IVS neuroblastoma patients that had enough tumor to determine the MYCN gene status, byMYCN protein expression, taking into account all samples obtained throughout the course of disease. Relapse-free survival was significantly differentfor 17 patients with detectable MYCN protein compared with 20 patients with undetectable MYCN protein (P = 0.0079) (A) and was not significantlydifferent for 14 patients with MYCN gene amplification compared with 23 patients without MYCN gene amplification (P = 0.35; median follow-up,7.3 years) (B). Two patients (one from each group) that died without relapse were treated as censored observations.


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tably, favorable initial chemotherapy responses of �90%

correlated only marginally with the MYCN protein content at

diagnosis (P = 0.053) but correlated strongly with its ex-

pression throughout the course of disease (P 0.00020).

Because overexpression of MYCN protein did not neces-

sarily correlate with gene amplification, we examined the out-

come in the 37 stage IVS, III, and IV patients that had sufficient

tumor for determining the MYCN gene status. At a median

follow-up of 7.3 years, the 20 patients with undetectable MYCN

protein before treatment clearly did better than the 17 with

overexpressed MYCN protein (55 versus 13% relapse-free, P

0.0079 in Fig. 3A; 46 versus 22% surviving, P = 0.030). This

was similarly seen for patients with undetectable compared with

overexpressed MYCN protein, taking into account all samples

obtained throughout the course of disease (73 versus 10% re-

lapse-free, P = 0.00039; 30 versus 14% surviving, P 0.0027).

In important contrast for this same group of 37 patients, al-

though 10 of the 14 with MYCN gene amplification rapidly

relapsed within 2 years and died quickly, 13 of the 23 without

MYCN gene amplification also relapsed subsequently, albeit

more slowly over 4 years and dying over 9 years. Thus, we

found no significant difference in outcome between patients

without or with MYCN gene amplification at 7.3 years of median

follow-up (43 versus 24% relapse-free, P = 0.35 in Fig. 3B; 39

versus 29% surviving, P 0.21); nor was such a difference

appreciable at 2 years of median follow-up (52 versus 39%

relapse-free, P = 0.57; 70 versus 43% surviving, P = 0.12).

That it might be the expression of MYCN protein per se

that is relevant for the clinical behavior of neuroblastoma is best

illustrated by the 23 patients known to have single-copy MYCN

gene. At a median follow-up of 6.8 years, the 17 stage IVS, III,

and IV patients with undetectable MYCN protein pretreatment

did significantly better than the 6 with overexpressed MYCN

protein (59 versus 0% relapse-free, P = 0.0016; 49 versus 17%

surviving, P = 0.022). This correlation also applies for patients

with undetectable compared with overexpressed MYCN protein,



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Fig. 4 Outcome of 23 stage IV, III, and IVS neuroblastoma patients known to be without MYCN gene amplification, by MYCN protein expression,taking into account all samples obtained throughout the course of disease. Kaplan-Meier curves of relapse-free survival (A) and overall survival (B).

showing a significant difference for 14 patients with detectable MYCN protein compared with 9 patients with undetectable MYCN protein (P -

0.00067 and 0.01 1 ; median follow-up, 6.8 years). One patient in the MYCN protein-negative group that died without relapse was treated as a censoredobservation; this explains why the relapse-free survival was 71%, whereas the overall survival was 67%.


taking into account all samples obtained throughout the course

of disease (Fig. 4, A and B).

Because conventional factors prognostic of neuroblas-

toma outcome were not uniformly distributed relative to the

expression of MYCN protein, it was important to stratify

them by log-rank analysis (Table 1). We found that stage and

ferritin significantly correlated with relapse, but histology

and VMA:HVA ratio did not, whereas age was of borderline

significance (Table 2). However, the significance of age and

ferritin was lost after stratifying for the effect of MYCN

protein, and the effect of stage became borderline. Similarly,

although stage, age, and ferritin significantly affected mor-

tality (P = 0.0050, 0.024, and 0.011, respectively) but his-

tology and VMA:HVA ratio did not (P = 0.47 and 0.19,

respectively), the significance of stage and age was lost after

stratifying for the effect of MYCN protein (P = 0.19 and

0.65, respectively), and the significance of ferritin became

borderline (P = 0.068). Tables 1 and 2 show the data for

MYCN protein expression during the course of disease; the

pretreatment expression of MYCN protein had a similarly

significant impact on prognosis. For example, the relapse rate

was higher for patients with detectable rather than undetect-

able pretreatment MYCN protein (P = 0.00014), despite

stratifying by age, stage, histology, or VMA:HVA ratio (P

0.0062, 0.0065, 0.00036, and 0.0034, respectively) but not by

ferritin (P = 0. 16), suggesting that overexpression of MYCN

protein might be inherent rather than acquired with treatment

and relapse.

Because amplification of the MYCN gene, an acknowl-

edged unfavorable factor, was not uniformly distributed relative

to MYCN protein expression during the course of disease, we

stratified it by log-rank analysis (Table 1). At a median fol-

low-up of 7.3 years, the prognosis was apparently not much

better without than with MYCN gene amplification, either before

(P = 0.35, 0.21) or after stratification of the comparisons of

relapse and mortality rates by the effect of MYCN protein (P =

0.39 and 1.00) or by the other conventional prognostic factors

(nonsignificant P values not shown). In contrast, the expression

Table I Neurobla stoma by prognostic factors and status of MYCNprotein or MYCN gene

MYCN proteindetectable throughout

the course of disease

No. of patients with +

known prognosticPrognostic factor factor No. % No. %

Age 57<lyr 16 53 5 19

�lyr l4 47 22 81

Histology 57

Favorable 12 40 2 7

Unfavorable I 8 60 25 93

VMA:HVA ratio 52�l 12 48 7 26

<1 13 52 20 74

Ferritin 39<150 p.g/liter 10 56 6 29

�l50 p.g/liter 8 44 15 71

Stage 57IVS 8 27 1 4

III 12 40 9 33

IV 10 33 17 63

MYCN gene 37

<3 copies 14 93 9 4/�3 copies I 7 13 59

MYCN protein 57Negative 30 100

Positive 27 100

of MYCN protein significantly correlated with the relapse rate,

both without and with stratification by each of the conventional

risk factors (Table 2). This correlation similarly applied for the

prognostic impact of pretreatment or posttreatment MYCN pro-

tein expression that had been stratified by the MYCN gene status

(P = 0.021 and 0.0007). Likewise, MYCN protein expression

remained prognostic when stratified simultaneously for stage

and age (P = 0.00076). Furthermore, the expression of MYCN

protein significantly affected the mortality rate (P = 0.00002)




Table 2 Outcome of neuroblastoma stratified by prognostic factors, M YCN protein and gene

Each prognostic factor MYCN protein throughout

Stratified by MYCNNot stratified protein

Stratified by prognosticfactor

Relative relapse rate Relative relapse rate Relative relapse rate

O:E O:E O:E

Prognostic factor ratio” P ratio” P ratio” P

Age<I yr�l yr

0.40 0.096 0.85 0.85

1.44 1.040.37 0.00011

1.80Histology

Favorable 0.65 0.36 1 .70 0.28 0.33 0.00001

Unfavorable 1. 12 0.92 2.17VMA:HVA ratio

�l 0.72 0.25 0.82 0.58 0.34 0.00001

<I 1.22 1.10 2.01

Femtin<150 xg/liter�l50 p.g/liter

0.56 0.031 0.64 0.13

1.53 1.330.49 0.013

1.56Stage

IVS 0.36 1.17 0.36

III 0.54 0.0015 0.50 0.051 0.00004

IV 1.88 1.50 1.89

MYCN gene<3 copies

�3 copies

0.83 0.35 1.22 0.39

1 .34 0.8 1

0.40 0.00070

1.44MYCN protein

Negative

Positive0.31 0.00001”

2.37

a O:E ratio is the ratio of observed relapses to the risk of relapse. P values are overall P values for the relative relapse rates of each prognostic

subgroup.

I, Overall P values for the relative relapse rates of MYCN protein-negative versus positive subgroups after simultaneously stratifying by stageand age (P = 0.00076).

despite stratification by stage, age, ferritin, VMA:HVA ratio, or

histology (P 0.0012, 0.0012, 0.027, 0.00059, and 0.00007,

respectively); by stage and age simultaneously (P 0.0077); or

by the MYCN gene status (P = 0.0059).

Because we could not study patients from 1964-1990

without pretreatment samples available for MYCN protein test-

ing, it was important to exclude the possibility of selection bias.

We report that the survival rates of study patients were not

significantly different from those of nonstudy patients or from

the overall survival rate for each of the nonlocalized stages (data

not shown).

DISCUSSION

Our data showed that MYCN protein that was heteroge-

neously expressed in neuroblastoma strongly correlated with

adverse outcome. MYCN protein detected in even a fraction of

the cells within the tumor also had prognostic significance (data

not shown). High MYCN mRNA levels had been observed

previously in neuroblastoma without MYCN gene amplification

(26, 27). Some clinical studies (28, 29), but not others (30, 31),

reported that high MYCN mRNA expression correlated with

poor outcome. The heterogeneity of MYCN protein expression

that we found in individual tumor cells might explain why

different pooled-cell MYCN mRNA studies had observed dis-

crepant results. Immunohistochemical evaluation of MYCN

protein in individual tumor cells may be used to circumvent this

clinical heterogeneity of MYCN expression. Furthermore, direct

measurement of the effector molecule is important, because

MYCN mRNA levels may not necessarily correlate with

MYCN protein levels. Ideally, MYCN mRNA determination by

Northern or reverse transcription-PCR analysis should be used

to corroborate the MYCN protein data, but these assays could

not be confidently performed with our available archival mate-

rial. Our results also differed from those of a previous study that

concluded that MYCN protein expression had no prognostic

significance. This difference might be due to technical reasons,

because we used two monoclonal antibodies specific for MYCN

protein that work in fixed tissues, whereas the previous study

used a polyclonal anti-MYCN antiserum in cryostat sections

(32).

We have shown that the expression of MYCN protein

correlated significantly with the long-term outcome, despite

stratification by each of the conventional prognostic factors,

including stage and age, and the MYCN gene amplification

status. This clinical evidence strongly supports the hypothesis

that the MYCN oncogene is pivotal for regulation of critical

cellular activities contributing to neuroblastoma progression

(12, 18, 19). Our MYCN gene data are also consistent with the

previous data showing that genomic amplification occurs mostly

in nonlocalized tumors, correlating with poor prognosis (1 2, 18,

19, 33), but rarely in localized tumors, in which it does not

necessarily correlate with adverse outcome (34). Therefore, our




data in fact corroborate rather than dispute the acknowledged

unfavorable impact of MYCN gene amplification in nonlocal-

ized neuroblastoma, besides offering a possible explanation for

the traditional observation that many nonamplified tumors also

ultimately fail therapy. Because the survival rates of study

patients did not differ significantly from those of nonstudy

patients or from the overall survival rate for each stage, the

MYCN findings in the cohort of patients that we have studied

are unlikely to be due to selection bias. However, because our

patient population is relatively small and has been treated over

a number of years, our observations require confirmation in a

larger number of prospectively studied patients in whom the

newer prognostic markers are available.

The amplified MYCN gene most likely acts through overex-

pression of MYCN protein. However, gene amplification is only

one way by which protein expression can be enhanced. We have

found, in stages III, IV, and IVS but rarely in stages I and II, that

MYCN protein overexpression can occur without gene amplifica-

tion (26% initially, 39% overall). Detectable MYCN protein reli-

ably predicted an unfavorable outcome in such nonlocalized tu-

mors, whereas the undetectable protein correlated with good

prognosis. Therefore, despite our small numbers of nonamplified

tumors, they best demonstrate that overexpression of MYCN pro-

tein per se is apparently sufficient for adversely affecting outcome.

This clinical finding is also inconsistent with the possibility that

some unknown genes or coamplified genes, rather than the MYCN

gene, are critical for progression of neuroblastoma.

This retrospective study does not allow further investigation

into the basis for MYCN protein overexpression in the absence of

gene amplification. However, a previous investigator has found

high MYCN mRNA levels associated with a long half-life MYCN

protein in a rapidly proliferating tumorigenic line established from

a progressive but nonamplified neuroblastoma (35). Another inves-

tigator has found high MYCN mRNA levels with increased protein

in two nonamplified tumorigenic lines, both of which proliferated

slower than MYCN gene-amplified lines (36). It remains to be

defined whether the expression of MYCN protein without gene

amplification is regulated at the transcriptional. translational, or

posttranslational level.

The 60-63-kDa MYCN nucleoprotein is essential during

embryogenesis (37). It subsequently becomes undetectable ex-

dept in neuroblastoma, retinoblastoma, Wilms tumor, and small-

cell lung or brain tumors (12, 18, 19), particularly the more

primitive tumors (29, 38). The MYC family proteins are tran-

scriptional regulators that play a pivotal role in regulating cel-

lular proliferation, differentiation, and apoptosis (39, 40). MYC

proteins interact with Max, another helix-loop-helix leucine

zipper protein, forming sequence-specific DNA-binding corn-

plexes that regulate the transcription of genes controlling the

cell cycle (40) and competing with alternative Max partners

Mad and Mxil to regulate differentiation (41-43). Because the

choice to undergo proliferation or differentiation may depend on

the level of MYCN expression, a more aggressive phenotype in

neuroblastoma might be conferred if the threshold of MYCN

protein-mediated transcription-regulatory activity is reached,

whether or not this is due to gene amplification. MYCN protein

might also mediate the transcription of genes that regulate the

response to chemotherapy. We have reported that P-glycopro-

tein expression correlated with poor prognosis, more primitive

disease, more advanced stages, and metastasis rather than the

primary neuroblastoma (15). Some studies of P-glycoprotein

mRNA (13, 14, 16), but not all (17, 29, 38), have agreed with

our findings; other studies of MRP mRNA have reported a

similar prognostic association (16, 17). Both P-glycoprotein and

MRP frequently coexpress with MYCN in neuroblastoma ( IS-

17, 29, 38), but it is unknown whether they affect prognosis

independently. If they do, this might suggest that each is integral

to malignant progression. Therefore, defining whether the ex-

pression of MYCN and drug resistance are regulated/coregu-

lated by humoral factors, cellular factors, cytokines, and biolog-

ical response modifiers or expressed/coexpressed with other

oncogenes, tumor suppressor genes, or flanking coamplified

genes might ultimately suggest a more effective therapy for this

highly fatal tumor.

ACKNOWLEDGMENTS

We acknowledge the contributions of Drs. H. Kahn. P. S. Thorner,

and S. S. Weitzman in reviewing the slides and patients and A. E. Evans

in reviewing the manuscript.

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1997;3:1699-1706. Clin Cancer Res H S Chan, B L Gallie, G DeBoer, et al. prognosis.MYCN protein expression as a predictor of neuroblastoma

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