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Molecular Oncology, Markers, Clinical Correlates |
Department of Pediatric Oncology, University Children’s Hospital, 50924 Köln, Germany
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ABSTRACT |
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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.
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Introduction |
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Some years ago Srivatsan et al. (5 , 6) determined LOH in chromosome arm 11q in 42% and 32% of the investigated cases, respectively, and concluded that "sequences" in this region might play a role in NB tumorigenesis. Other workers confirmed frequent deletions in 11q after LOH studies with a lower (7 , 8) or higher frequency (9 , 10) . Allelic loss in chromosome 3p was first described by Hallstensson et al. (11) . Since 1997 several investigations using CGH found loss of chromosomal material at distal 11q and at 3p recurrently, and in advanced NB (12, 13, 14, 15, 16, 17, 18) . To date, however, the prognostic influence of these aberrations remains unclear. Besides their frequency, alterations in chromosomes 3 and 11 are of special interest as new prognostic parameters because recent studies could determine an inverse association between LOH 11q and/or LOH 3p and MNA (10 , 17) .
The aim of this study was to specify the frequency and type of alterations in distal chromosome arm 3p and 11q in a large collective of NB tumors. Using FISH as an alternative technique to LOH studies, we further investigated the relation between 3p and 11q aberrations, on the one hand, and changes in 1p36 and amplification of MYCN, on the other hand, in single cells of the same tumor. Finally, we analyzed the influence of 3p and 11q alterations on the patient’s outcome.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Alterations in chromosome arms 3p and 11q were analyzed using interphase FISH in a dual-color procedure. For chromosome 3 we applied DNA probes 3ptel (D3S4559) and D3Z1 (centromere; Oncor, Gaithersburg, MD), and for chromosome 11, 11q23 (MLL) and D11Z1 (centromere). DNA probes 3ptel and MLL are both located in the commonly deleted region, as determined by CGH data. Centromere probes served as hybridization controls and were used to determine the copy number of chromosomes 3 and 11.
Similar to the classification of the European Neuroblastoma Quality Group for chromosome 1p alterations, we distinguished two types of alterations:
(a) Deletion 3p (11q): only one copy of 3p26 (11q23) but at least two centromere copies [monosomy 3p26 (11q23)] 
ratio centromere copies to 3p26 (11q23): 2:1, 3:1, and so on.
(b) Imbalance 3p (11q): at least two copies of 3p26 (11q23) with additional centromere copies ratio centromere copies to 3p26 (11q23): 3:2; 4:2, 4:3, and so on.
Additionally, 142 of 144 tumors in the collective were investigated for a possible amplification of the MYCN oncogene using FISH probes n-myc and D2Z (Oncor). One hundred thirty-nine of 144 cases were analyzed for alterations in chromosome 1p36 using D1Z1 in 1q12 and D1Z2 in 1p36.33 (p1–79; American Type Culture Collection, Manassas, VA). MNA was defined as at least the 10-fold number of MYCN copies compared with the copy number of chromosome 2; alterations in chromosome arm 1p were classified as mentioned above. Between 100 and 465 nuclei (mean 110, for chromosomes 3 and 11, and mean 200 for chromosome 1 and MYCN) were counted in one tissue sample in each case.
Statistical Analyses.
To compare variables of interest, Fisher’s exact test, 
2 test, or Mann-Whitney t test were used where appropriate. Kaplan-Meier estimates for EFS were calculated and compared by log-rank test. For multivariate analysis, Cox’s proportional hazards regression model built on EFS was used.
Recurrence, progression of disease, and death from disease were counted as events. Death resulting from therapy complications was not counted as an event, but censored for EFS analysis.
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Results |
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. Amplification of the MYCN oncogene was detected in 4 of 41 (10%) patients with aberrations in 11q compared with 21 of 100 (21%) in the 11q normal cases. Thus, loss of chromosomal material in 11q and MNA tended to show an inverse correlation (P = 0.07) but did not mutually exclude each other. Changes in chromosome arm 1p were found in 10 of 39 (26%) of the investigated samples with 11q aberrations [5 of 27 (19%) with 11q deletion and 5 of 12 (42%) with 11q imbalance]. In the 11q normal cases 1p alterations occurred in the same percentage, 26 of 100 (26%), therefore, an inverse correlation could not be delineated. Children with 11q deletion were older (median age, 4.0 years) than patients with 11q imbalance (median age, 2.6 years) and those with normal 11q (median age, 1.2 years; P < 0.001).
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Altogether, one-third (n = 48, 33%) of all investigated tumors in our cohort showed an alteration 11q and/or 3p (Table 1)
. The aberrations were highly associated with each other (P < 0.001). Fifteen tumors showed deletions in both chromosomes, three more imbalances in 3p and 11q (one of them also in 1p). Of the cases with 11q alterations, 20 of 42 (48%) showed a deletion or imbalance 3p as well. In the 3p aberrant sample, 20 of 26 (77%) displayed changes in 11q simultaneously.
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). This, however, was not true for the patients with stage 4 disease: 3-year EFS was 40 ± 17% and 33 ± 27% for patients with deletion and imbalance 11q and 30 ± 12% for those with no alteration (P = 0.84). However, in the subgroup of favorable stages (localized and 4s), 11q status could discriminate between good and bad prognosis with strong statistical significance (3-year EFS of patients with 11q alterations was 43 ± 17% versus 71 ± 7% for patients with normal 11q; P = 0.0027), especially in MYCN nonamplified tumors (3-year EFS: 43 ± 18% versus 84 ± 6%, P = 0.0006; Fig. 2B
). Of 12 patients with 11q alterations, favorable stages, and no MNA, 6 experienced progression of disease, 5 of them before 24 months after diagnosis (Table 1)
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). Four patients with 3p imbalance have not experienced an event up to now after a follow-up of between 6 and 62 months. The outcome of patients with 3p deletion (3-years EFS: 13 ± 12%, n = 22) was worse than that of patients with a normal 3p (3-year EFS: 62 ± 6%, n = 118, P = 0.01). Similar to the results for 11q, patients with stage 4 disease and 3p alterations (3-year EFS: 39 ± 17%, n = 20) showed a poor outcome similar to those with stage 4 but no 3p alteration (3-year EFS: 31 ± 11%, n = 39). In contrast, patients with favorable stages and aberrations in 3p (3-year EFS: 0 ± 0%, n = 7) had a significantly worse outcome compared with patients with favorable disease and normal 3p (3-year EFS: 77 ± 6%, n = 79, P = 0.002). In the subgroup of patients with favorable disease and no MNA, a significant difference in outcome was found as well when 3p was considered (3-year EFS of 5 patients with 3p alterations was 0 ± 0% versus 76 ± 6% for 76 patients without 3p aberrations, P < 0.0001; Fig. 2D
). All four patients with 3p deletions in localized stages showed an event within 24 months.
The three chromosomal parameters—11q status (alteration versus no alteration), 3p status (alteration versus no alteration), and MYCN status (amplification versus no amplification)—were evaluated in a multivariate Cox regression model built on EFS. MYCN status (P < 0.001, exp(ß): 4.8) as well as 11q changes (P = 0.05, exp(ß): 2.4) but not 3p alterations were shown to be significant prognostic factors. Fig. 3
shows the EFS of four risk groups characterized by the MYCN status and the 11q status.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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A portion of 36% with LOH 11q was described by Luttikhuis et al. (20) in a sample of 28 patients (43% stage 4). Several other studies reported frequencies of LOH 11q between 19% and 32% (6, 7, 8) . CGH analyses detected deletions in 11q in 10–41% (12, 13, 14, 15, 16 , 18) . In a recent CGH study (17) , loss of material in 11q was determined in 59% of all stage 4 tumors, which is comparable with our results (47%). In contrast to disseminated NB, 11q alterations are far less common in locoregional and 4s tumors. In our sample, 9% of stages 1–3 and 4s tumors showed deletions and 7% imbalances. Other workers could report the presence of 11q loss in locoregional and 4s tumors in a similar order of magnitude (19%) (20) .
The second most frequent aberration in our sample was a deletion in chromosome band 3p26, found in 22 of 144 tumors (15%). Four more cases showed an imbalance 3p (3%), so that changes in 3p could be detected in 18% of primary NB tumors. Ejeskar et al. (21) found allelic loss in chromosome arm 3p in 15% of the analyzed tumors with the commonly deleted consensus region proximal band 3p26. In mainly favorable tumors, 3p loss was detected in 16% of the investigated NB samples (11) . Several CGH investigations expand the region of chromosomal loss to terminal 3p with frequencies of 13–25% (13 , 15, 16, 17) .
Besides the frequency of 3p alteration there is another consistent finding between our study and others. Changes in 3p can be found in 48% (20 of 42) of the cases with 11q aberrations; even larger (77%, 20 of 26) is the portion of 11q alterations in the 3p deleted and imbalanced group. The association between these aberrations is not as close as between 1p deletion and MNA but is confirmed by several studies (20 , 17) . To date, no functional relation between these aberrations is known. Guo et al. (9) suggested that 11q aberrations are late events in tumorigenesis because of the association between survival and 11q deletion. If so, aberrations in chromosome arm 3p are probably even later events because 3p aberrations without 11q changes are much rarer than vice versa. Another reason for this hypothesis is the fact that patients with 11q and 3p changes are significantly older compared with patients with normal 11q and 3p. These aberrations as well as imbalances of chromosome arm 1p36 (22) are characteristic features of progressive NB.
One of the most interesting findings is the rare occurrence of 1p alterations and MNA together with 11q and 3p changes, which is supported by several groups (9 , 10 , 17) . Although 11q aberrations were not exclusively found in MYCN nonamplified tumors (20) , they were rare in amplified cases (3 of 22, 14%). Corresponding to this, MNA was found in only 12% and 10% of cases with alterations in 3p and 11q. Altogether, 13% and 26% in the 3p and 11q aberrant cohort displayed deletions in 1p36. In agreement with other studies, an inverse association between 11q and MNA was found in stage 4 tumors (17) , however, not in localized and 4s disease.
Tumor cells that lose chromosomal material in 1p36 or develop an excess of MYCN copies in a later period of tumorigenesis are believed to show more aggressive growth, which explains the effect on the patient’s prognosis. Cells with 11q and 3p alterations probably display a similar pattern concerning growth and the patient’s outcome. Given that chromosome mutations in tumor cells occur coincidentally there is no reasonable explanation for the frequent changes in chromosomes 11 and 3 not occurring in 1p-deleted or amplified tumors. Independent of which aberration occurs earlier or later in tumor progression, it seems that the coexistence of 1p alterations/MNA and 11q/3p alteration cancel the growth advantage of the single aberration. The simultaneous loss of possible (tumor suppressor) genes on chromosome arms 1p, 3p, and 11q as well as an MYCN excess might cause a severe cell cycle damage, leading to cell death. Corresponding to this is an interesting observation that only 5 of 27 11q-deleted cases but 5 of 12 11q imbalances showed a deletion (n = 1) and imbalance (n = 4) in 1p simultaneously. Although the results show no significance yet, we would suggest that 11q imbalance cases are more tolerant against the effect of 1p alterations compared with the monosomic 11q deletions. Thus far, it is totally unclear which genes in 1p, 3p, and 11q cause the unfavorable effect in NB.
Patient Outcome.
Probably because of the short observation time of several patients, overall survival of all investigated cases after 3 years was high (83 ± 4%) whereas EFS was much lower (56 ± 6%). Patients with 11q alterations of all stages showed a significant poorer outcome compared with those without changes in chromosome 11. Prognosis was unfavorable for tumors with deletions as well as imbalances, which corresponds with recent observations on chromosome 1p36 alterations (22)
. In locoregional and stage 4s NB tumors, the outcome of patients with 11q aberrations was inferior on the whole as well for the nonamplified cases (P < 0.001).
Tumors of all stages with deletions in 3p displayed a poor outcome, too, whereas the patients with 3p imbalance cases did not show any event up to now. The EFS in stage 4 tumors was poor independent of 3p alterations. But similar to the 11q changes, the outcome for patients with 3p alterations was also worse in the subgroup of favorable stages (P = 0.0024) as well as in the subgroup of MYCN nonamplified favorable stages (P < 0.001).
In agreement with our present results, Plantaz et al. (17) described no difference in EFS between stage 4 patients with and without 11q LOH. Another study suggested a significantly poor EFS of MYCN nonamplified tumors with 11q LOH (20) .
The multivariate analysis including the chromosomal parameters MNA, 3p, and 11q alteration confirmed the importance of a MNA as a prognostic factor but also provided evidence for chromosomal changes in 11q as new reliable parameters to assess the patient’s outcome.
To summarize, we have presented the first FISH analyses on a large number of cases clearly indicating that aberrations in chromosome arms 3p and 11q are nonrandom changes especially in advanced stage tumors but also in localized and stage 4s cases. Their prognostic influence is significant in locoregional and stage 4s NB. The analysis on a single cell level allows distinction between deletion events, leading to a monosomy and those where the disomic situation remains. As shown for alterations in 1p36 (22) , both groups do not necessarily reveal the same associations to other molecular parameters or the same prognostic influence. Additional studies of larger patient numbers will clear the relevance of each aberration.
We conclude that loss of chromosomal material in chromosome arms 3p and 11q define new high-risk subgroups of NB, especially in MYCN single copy localized and stage 4s disease. Because of the close correlation of 3p and 11q abnormalities, the 11q investigation may, together with MYCN analyses, suffice for prognosis estimation particularly in localized stages.
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FOOTNOTES |
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1 Supported by The Fördergesellschaft Kinderkrebs-Neuroblastom-Forschung e.V. 
2 To whom requests for reprints should be addressed, at Department of Pediatric Oncology, University Children’s Hospital, Joseph-Stelzmann-Str. 9, 50924 Köln, Germany. Phone: 49-221-478-6816; Fax: 49-221-478-4689; E-mail: Ruediger.Spitz{at}medizin.uni-koeln.de
3 The abbreviations used are: NB, neuroblastoma; MNA, MYCN amplification; LOH, loss of heterozygosity; CGH, comparative genomic hybridization; FISH, fluorescence in situ hybridization; EFS, event-free survival.
Received 4/ 3/02; revised 6/ 5/02; accepted 7/15/02.
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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