The excellent prognosis of localized neuroblastoma in infants, the overdiagnosis observed in neuroblastoma screening studies, and several case reports of regression of localized neuroblastoma ...prompted us to initiate a prospective cooperative trial on observation of localized neuroblastoma without cytotoxic treatment.
For infants with localized neuroblastoma without MYCN amplification, chemotherapy was scheduled only in cases with threatening symptoms; otherwise, the tumor was either resected or observed by ultrasound and magnetic resonance imaging (MRI).
Of 340 eligible participants, 190 underwent resection, 57 were treated with chemotherapy, and 93 were observed with gross residual tumor. Of those 93 patients with unresected tumors, spontaneous regression was seen in 44, local progression in 28, progression to stage 4S in seven, and progression to stage 4 in four. Time to regression was quite variable, with first signs of regression noted 1 to 18 months after diagnosis and in 15 of 44 patients even after the first year of life. So far, complete regression was observed in 17 of 44 patients 4 to 20 months after diagnosis. Known clinical risk factors were not able to differentiate between patients with regression and regional or metastatic progression. Overall survival (OS; 3-year OS, 0.99 +/- 0.01) and metastases-free survival (rate at 3 years, 0.94 +/- 0.03) for patients with unresected tumors was excellent and was not different from patients treated with surgery or chemotherapy.
Spontaneous regression is regularly seen in infants with localized neuroblastoma and is not limited to the first year of life. A wait-and-see strategy is justified in those patients.
Purpose: To improve risk prediction in neuroblastoma and to specify the type of a possible relapse, alterations in the long arm of
chromosome 11 were analyzed.
Experimental Design: A representative ...cohort of 611 neuroblastomas was investigated for deletion events in distal chromosome 11q using interphase
fluorescence in situ hybridization.
Results: Alterations in 11q were found in 159 of 611 tumors in the whole cohort (26%) and were associated with stage 4 disease ( P < 0.001) and age at diagnosis of >2.5 years ( P < 0.001). Event-free survival and overall survival were significantly poorer for patients with 11q loss in the whole cohort
(event-free survival and overall survival, P < 0.001) and in different subsets: neuroblastoma without MYCN amplification (MNA) (event-free survival and overall survival, P < 0.001), with MNA (event-free survival, P = 0.03; overall survival, P = 0.02), and MYCN -nonamplified stage 1, 2, 3, and 4S tumors with and without del 1p (event-free survival and overall survival, P < 0.001). In stage 4, the 11q status did not discriminate outcome. By multivariate analysis, the 11q status proved prognostic
for event-free survival in the whole cohort ( P = 0.008; hazard ratio, 1.573) and in the subgroup of stages 1, 2, 3, and 4S without MNA ( P < 0.001; hazard ratio, 3.534). Moreover, 11q alterations were strongly correlated with the occurrence of metastatic relapses
( P < 0.001).
Conclusion: In addition to the current risk stratification, the status of 11q enables the identification of patients with an increased
risk for relapses in general and metastatic relapses in particular.
Purpose: MYCN amplification is an important therapy-stratifying marker in neuroblastoma. Fluorescence in situ hybridization with signal detection on the single-cell level allows a critical judgement ...of MYCN intratumoral heterogeneity.
Experimental Design: The MYCN status was investigated by fluorescence in situ hybridization at diagnosis and relapse. Heterogeneity was defined as the simultaneous presence of amplified cells (â¥5 cells
per slide) and nonamplified cells within one tumor or sequential change of the amplification status during the course of the
disease. Likewise, heterogeneity can be detected between primary tumor and metastasis.
Results: From 1,341 patients analyzed, 1,071 showed no amplification, 250 showed homogeneous amplification, and 20 patients showed
MYCN heterogeneity. Of the patients with heterogeneity, 12 of 20 had clusters of MYCN amplifications, 3 of 20 had amplified single cells, 3 of 20 showed MYCN amplifications in the bone marrow but not in the primary tumor, and 2 of 20 acquired MYCN amplification during the course of the disease. All stage 4 patients were treated according to high-risk protocols; 7 of
8 later progressed. Four patients with localized disease were treated according to high-risk protocol because of MYCN -amplified clusters; 1 of 4 later progressed. One patient treated with mild chemotherapy experienced progression. Seven patients
with localized/4S disease underwent no chemotherapy: 4 of 5 patients with MYCN heterogeneity at diagnosis remained disease-free, and 1 of 5 experienced local progression. Two patients had normal MYCN status at diagnosis but acquired MYCN amplification during the course of the disease.
Conclusion: MYCN heterogeneity is rare. Our results suggest that small amounts of MYCN -amplified cells are not correlated to adverse outcomes. More patients with heterogeneity are warranted to clarify the role
of MYCN heterogeneity for risk classification.
Purpose: To find new nonrandom chromosomal changes in neuroblastoma (NB) with a potential to forecast the patient’s outcome, alterations
in chromosome arms 3p and 11q were investigated.
Experimental ...Design: Frequency and prognostic potential of 3p and 11q alterations in 144 NBs were analyzed using interphase fluorescence in situ hybridization with DNA probes for 3p26 and 11q23. Aberrations were defined as deletion (monosomy of a specific region) or
imbalance (at least two intact and additional 3p26- or 11q23-deleted chromosomes).
Results: Forty-two of 144 cases (29%) displayed 11q alterations (21% deletions, 8% imbalances). Most aberrations were associated with
stage 4 disease (28 of 59, 47%) but were also present in localized and 4s tumors (14 of 85, 16%; P = 0.007). Patients with 11q deletion/imbalance were significantly older at diagnosis ( P < 0.001). Changes in 3p were detected in 26 of 144 (18%) samples (15% deletions, 3% imbalances). These alterations were also
associated with stage 4 20 of 59 (34%) versus 6 of 85 (7%) in stages 1–3 and 4s, P = 0.007, and the median age was increased ( P < 0.001). Aberrations in both chromosomes were highly associated with each other ( P < 0.001). MYCN amplification (MNA) was detected in 10% and 12% of tumors with 11q and 3p alterations, and changes in 1p36 occurred in 13%
and 26% of the 3p- and 11q-aberrant tumors. MYCN amplification and 11q deletion/imbalance tended to show an inverse correlation ( P = 0.07) as well as 1p and 3p deletion/imbalance ( P = 0.07). Patients with 3p and 11q abnormalities in localized/4s tumors showed an inferior outcome compared with those without
these alterations ( P = 0.002 and P = 0.0027, respectively), in particular in MYCN single copy tumors ( P < 0.0001 and P = 0.0006, respectively).
Conclusion: Alterations in 3p and 11q are frequent nonrandom aberrations in NB and define a new high-risk subgroup in MYCN single copy stage 1–3 and 4s disease.
Purpose: In several studies, gain of the distal long arm of chromosome 17 was shown to be a frequent and prognostically relevant factor
in neuroblastoma, in addition to MYCN amplification (MNA) or 1p ...deletion. We asked whether this observation could be confirmed in a German cohort.
Experimental Design: To evaluate the frequency and prognostic impact of 17q gain, we investigated tissue samples from 193 neuroblastoma patients
by the use of fluorescence in situ hybridization. The DNA probe ( MPO ) was located in distal 17q in the region of interest as used by several groups. To analyze the association of patients’ outcome
with the breakpoint position within 17q, we used the more proximal DNA probe ERBB2 in 17q21 on a selected number of cases. Gain was defined as an excess of 17q material compared with the chromosome 17 centromere
in at least 50% of the analyzed tumor cells. In addition, alterations in chromosomes 1p, 3p, and 11q, as well as MYCN status, were determined to describe the interrelationship between the different parameters and to evaluate an independent
prognostic influence.
Results: Gain of 17q was found in 61% of the investigated tumors. An additional 23% displayed an excess of 17q in less than half of
all cells. Gain correlated with stage 4 disease ( P = 0.003) and with other chromosomal alterations, such as 1p ( P < 0.001), 3p ( P = 0.01), 11q ( P = 0.001), and MNA ( P = 0.016), as well as with increased patient age ( P = 0.01). Outcome was not different between patients with 17q gain compared with those without, however. A prognostic influence
could not be delineated in all stages or in localized or in stage 4 subgroups or in the MYCN nonamplified patient cohort. Outcome did not differ between patients with additional 17q material in <10% of the cells or
in >70%. Patients showing a breakpoint in the more proximal part of 17q could not be described as having a more favorable
outcome compared with patients with a more distal breakpoint. Prognosis was poor in patients with MNA and/or 11q loss either
with or without 17q gain. A multivariate analysis including the chromosomal parameters 17q, 11q, and MYCN status, as well as stage, showed MYCN and 11q status ( P < 0.001), but not 17q or stage as significant prognostic factors.
Conclusion: Although the most frequent aberration in neuroblastoma to date, we found no association between 17q gain and an inferior
prognosis in our cohort; the status of MYCN and 11q, however, allowed reliable prediction of patients’ outcomes.
To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease.
Two hundred fifty-one neuroblastoma specimens were analyzed using a customized ...oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States.
The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival EFS 0.86 +/- 0.03 favorable; n = 115 v 0.52 +/- 0.07 unfavorable; n = 59 and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P < .0001) and separated risk groups of current neuroblastoma trials into subgroups with divergent outcome (NB2004: low-risk 3-year EFS 0.86 +/- 0.04 v 0.25 +/- 0.15, P < .0001; intermediate-risk 1.00 v 0.57 +/- 0.19, P = .018; high-risk 0.81 +/- 0.10 v 0.56 +/- 0.08, P = .06). In a multivariate Cox regression model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 95% CI, 2.544 to 8.893).
Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.
Purpose: The tumor-associated antigen PRAME , a potential candidate for immunotherapeutic targeting, is frequently expressed in a variety of cancers. However, no information
about its presence in ...neuroblastoma is available to date. We therefore evaluated and quantified PRAME expression in a considerable number of neuroblastoma tumors and assessed its impact on the outcome of patients.
Experimental Design: Qualitative analysis of PRAME expression was assessed by reverse transcription (RT)-PCR screening of 94 patients with primary neuroblastoma. The same cohort
was used for semiquantitative determination of transcript levels by Northern blotting, comparing the signal intensities of
patients with those of testis total RNA. For more precise quantification of PRAME expression, real-time RT-PCR was performed in 88 patients of the above cohort and 7 additional patients, thus leaving a total
of 101 patients that were analyzed with either method. Furthermore, association with tumor stage, age of patients at diagnosis,
and MYCN amplification was determined as well as the prognostic impact of PRAME expression.
Results: RT-PCR screening detected PRAME expression in 93% of primary neuroblastoma and 100% of patients with advanced disease. Furthermore, RT-PCR and Northern blot
analysis showed a highly significant association of PRAME expression with both higher tumor stage ( P < 0.01) and the age of patients at diagnosis ( P < 0.01). Finally, precise quantification of PRAME expression by quantitative real-time reverse transcription-PCR displayed significant impact on the outcome of patients.
Conclusions: PRAME expression in neuroblastoma is extraordinarily common and was universally seen in patients with advanced-stage disease in
our study. Furthermore, significant impact of PRAME expression on the outcome of patients was shown. Thus, PRAME may present a particularly attractive target for immunotherapeutic strategies in neuroblastoma.
While the role of
MYCN-amplification (MNA) for risk assessment in neuroblastoma is undisputed, the phenomenon of gene copy excess below the amplification threshold is rarely described. To discuss ...biological characteristics and the clinical impact of the so- called
MYCN-gain
versus amplified or non-amplified cases, we investigated the
MYCN status of 659 patients uniformly analysed by fluorescence
in situ hybridisation. The number of
MYCN-amplified tumours in our cohort was 18% (116/659); an additional 38 tumours (6%) displayed
MYCN-gain. Both alterations were associated with an advanced stage disease, an increased patient age and further chromosomal alterations. Most of the amplified neuroblastomas displayed 1p aberrations, whereas
MYCN-gain tumours correlated with 11q alterations. In contrast to the amplified cases, tumours with gain displayed no increased
MYCN RNA levels. MNA
versus non-amplification discriminated between good and poor outcomes, independent of stage, age and the degree of amplification. However, patients with amplified tumours showed a significantly better outcome when this was combined with non-stage 4 disease and age <1 year
versus stage 4 and age >1 year. Although
MYCN-gain was associated with poor event-free-survival (EFS) in stages 1–3, 4S (
P=0.005), this might be related to associated genetic aberrations and not to the
MYCN-gain itself. A survival difference between neuroblastomas with gain and single copy
MYCN could not be delineated. In conclusion, MNA predicts a poor outcome for neuroblastoma patients of all stages and age.
MYCN-gain is also a characteristic feature of advanced stage tumours and older patients, but is not associated with higher
MYCN expression and appears not to be discriminative in predicting patient outcome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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