Abstract
Background
High-level MYCN amplification (MNA) is associated with poor outcome and unfavorable clinical and biological features in neuroblastoma. Less is known about these associations in patients with low-level MYCN copy number increases.
Methods
In this retrospective study, we defined patients as having tumors with MYCN wild-type, MYCN gain (2–4 fold increase in MYCN signal compared to reference probe), or MNA (>4 fold increase). We used tests of trend to investigate ordered associations between MYCN copy number category and features of interest. Log-rank tests and Cox models compared event-free (EFS) and overall survival (OS) by subgroup.
Results
Among 4,672 patients, 3,694 (79.1%) had MYCN wild-type tumors, 133 (2.8%) had MYCN gain, and 845 (18.1%) had MNA. For each clinical/biological feature, the proportion of patients with an unfavorable feature was lowest in the MYCN wild-type category, intermediate in the MYCN gain category, and highest in the MNA category (p<0.0001), except for 11q aberration where the highest rates were in the MYCN gain category. Patients with MYCN gain had inferior EFS and OS compared to wild-type. Among patients with high-risk disease, MYCN gain was associated with the lowest response rate following chemotherapy. Patients with non-stage 4 disease and patients with non-high risk disease with MYCN gain had significantly increased risk for death, a finding confirmed on multivariable testing.
Conclusions
Increasing MYCN copy number is associated with an increasingly higher rate of unfavorable clinical/biological features, with 11q aberration an exception. Patients with MYCN gain have inferior outcomes, especially in otherwise more favorable groups.
Keywords: MYCN, amplification, gain, neuroblastoma, prognosis, segmental chromosomal aberration, ploidy
Introduction
Neuroblastoma, the most common extra-cranial solid tumor affecting children, is remarkable for its extreme clinical heterogeneity. Although the 5-year overall survival (OS) has increased over the past several decades, generalizing OS for all neuroblastoma patients can be misleading. Studies of several known prognostic factors in neuroblastoma have revealed that some have a greater impact on outcome than others, and some are only prognostic in subsets of patients.1–4 Most notably, MYCN gene amplification (MNA) has proven to be an independent prognostic factor for identifying rapid tumor progression and predicting poor prognosis irrespective of age and clinical stage.5,6 MNA, which occurs in about 16% of cases of neuroblastoma,7 was one of the first tumor-derived genetic markers that was shown to be of clinical and prognostic value. Current guidelines for the assignment of risk group used by the International Neuroblastoma Risk Group (INRG) and the Children’s Oncology Group (COG) heavily weigh MYCN amplification.1,8
In addition to established correlation with prognosis, MNA is also known to be associated with important clinical and biological features.7 MNA is more common in adrenal primary tumors and less common in thoracic primary tumors.4,7 There is strong association between MNA and other tumor biologic and genomic features, such as unfavorable histology,9,10 diploidy,11–13 high mitotic karyorrhectic index (MKI),14 and loss of heterozygosity at 1p.15–19
MYCN copy number is a discrete variable, but groups have applied cut-points that define specific tumors as amplified vs. non-amplified. In the COG, MNA is defined using fluorescence in situ hybridization (FISH) as greater than a 4-fold increase in MYCN signal number compared to centromeric reference probe, a definition that is consistent with international consensus but without a clear biological rationale for an exact threshold of 4-fold.20 The COG further categorizes MYCN copy number into 4 groups: wild type (less than 2 fold increase in MYCN signal); MYCN gain (2–4 fold increase); low-level MNA (5–10 fold increase); and high-level MNA (>10 fold increase). Likewise, the SIOPEN group classifies tumors with 2–4 fold increase as “MYCN gain (inconclusive)”.21
The majority of cases with >2 fold increase in MYCN signal have high-level MNA (> 10 fold increase), thus less is known about patients with MYCN gain or low-level MNA. Two prior studies reported that MYCN gain was associated with inferior outcomes compared to patients with wild-type tumors,22,23 though a third study reported superior outcomes for this group.24 Interestingly, MYCN gain appears to be positively associated with 11q aberration, whereas MNA is inversely associated with 11q aberration, suggesting that the relationship between MYCN copy number with other clinical or biological features may be complex.22,23
To understand the prognostic impact of MYCN copy number and provide new insights into the relationship between MYCN copy number and other clinical and biological features of neuroblastoma, we conducted a comprehensive analysis of a large cohort of patients with known MYCN copy number. We also attempted to determine if the relationship of MYCN copy number with these clinical and biological features demonstrated trends with increasing copy number.
Patients & Methods
Patients
Patients were eligible for inclusion in the analytic cohort if they enrolled in the COG Neuroblastoma Biology study ANBL00B1 from 11/1/2007–2/28/2016 prior to treatment, had a confirmed diagnosis of neuroblastoma or ganglioneuroblastoma (intermixed or nodular), and had satisfactory centralized test results reported for MYCN. Consent was obtained at time of enrollment to ANBL00B1. This retrospective analysis was deemed exempt from further review by the Dana-Farber Cancer Institute Office for Human Research Studies.
Primary Predictor Variable
Our primary predictor variable was MYCN copy number category. MYCN copy number was determined by interphase fluorescence in situ hybridization (FISH) performed in the Neuroblastoma Reference Laboratory at Nationwide Children’s Hospital using frozen or paraffin-embedded tumor tissue or bone marrow with confirmed neuroblastoma cells. FISH was performed using a dual color probe (Abbott Molecular), with a CEP2 centromeric probe as reference. MYCN wild type was defined as less than 2 fold increase in MYCN signal and MYCN gain as 2–4 fold signal gain in ≥ 20% of cells compared to the reference probe. MNA was defined as greater than a 4 fold increase in MYCN signal compared to a reference probe, with low-level MNA defined as 5–10 fold increase in signal and high-level MNA as > 10 fold increase in signal. As the focus of this report is on MYCN copy number, heterogeneous MYCN amplification (presence of MNA in < 20% of tumor cell nuclei) was not evaluated further in this study.
Outcome Variables
Clinical variables of interest included sex, age, International Neuroblastoma Staging System (INSS) stage,25 COG risk group,26 primary site, LDH level (dichotomized at group median), and ferritin level (dichotomized at group median). Biological variables of interest, determined centrally by the Neuroblastoma Reference Laboratory and pathologists, included ploidy (hyperdiploidy=any DNA index>1.0), 1p loss of heterozygosity (LOH), 11q aberration (unbalanced LOH27), INPC histologic classification,10 mitosis karrhyohexis index (MKI), and grade of differentiation. Clinical outcomes were end-induction response to chemotherapy, event-free survival (EFS), and overall survival (OS). Response to induction chemotherapy was defined using the International Neuroblastoma Response Criteria25 and was only available for patients with high-risk disease treated on COG protocols ANBL053228 and ANBL12P1.29 For patients with MIBG-avid disease, MIBG imaging was a standard component of response assessment. EFS was defined as time from diagnosis to first episode of relapse, progression, death, or secondary malignancy; patients without an event were censored at date of last contact. OS was defined as time from diagnosis to death; surviving patients were censored at date of last contact.
Statistical Analyses
Associations between MYCN copy number categories and clinical and biological features were assessed using Cochran-Armitage tests for trend,30 testing the null hypothesis of no linear trend against an alternative of a linear trend, with at least one strict inequality between groups. Chi-squared tests or Fisher’s exact tests were used to conduct two group comparisons between clinical and biological features.
Tests of association between end-induction response and MYCN copy number category were performed using a Cochran-Armitage test for trend. EFS and OS were estimated per Kaplan-Meier and survival curves compared between MYCN copy number categories using log-rank tests.
Cox proportional hazards models tested the prognostic strength of MYCN copy number category in a univariate model and in multivariable models using standard neuroblastoma risk factors (age, INSS stage, ploidy, MKI, and grade of differentiation). We repeated analyses within the following subgroups of interest: high-risk, INSS Stage 4; high-risk, regardless of stage; stage other than Stage 4; and non-high-risk. Tests for violations of the proportional hazards (PH) were performed. If the PH assumption was violated, a time-dependent covariate was included in the model in the model. In these cases, p-values from time-dependent covariate adjusted Cox models are presented.
Results
Patient Characteristics and Distribution of MYCN Copy Number
Among 5,176 patients with neuroblastoma or ganglioneuroblastoma who enrolled prior to treatment on COG Neuroblastoma Biology study ANBL00B1 from 11/1/2007–2/28/2016, 4,672 patients (90.3%) had available MYCN copy number data and form the analytical cohort for this report. Details of the 504 patients with unknown MYCN status are shown in Supplemental Table 1.
Of the 4,672 patients in the analytical cohort, 3,694 (79.1%) had MYCN wild-type tumors, 133 (2.8%) had tumors with MYCN gain, 10 (0.2%) had low-level MNA, and 835 (17.9%) had high-level MNA. Due to the small number of patients with low-level MNA, low-level and high-level MNA were combined into one MNA group for remaining analyses (see Supplemental Table 2 for details of the 10 patients with low-level MNA).
The characteristics of the overall cohort (Table 1) appear to be representative of the neuroblastoma population, with a slight male predominance and median age of 20.7 months [range: −4 days (diagnosis in utero) to 30.1 years].
Table 1.
Clinical and biological features according to MYCN copy number in patients with neuroblastoma (entire cohort: n = 4,672).*
| Feature | All Patients N (%) |
A: MYCN Wild-Type N (%) |
B: MYCN Gain (signal 2–4x) N (%) |
C: MYCN Amp (signal >4x) N (%) |
p-value(A vs. B vs. C) | p-value for Non-Amplified Groups(A vs. B) | p-value for Non-Wild-Type Groups(B vs. C) |
|---|---|---|---|---|---|---|---|
|
| |||||||
| Clinical Features | |||||||
|
| |||||||
| Male | 2,456 (53) | 1,878 (51) | 75 (56) | 503 (60) | <0.0001 | 0.2083 | 0.4942 |
| Female | 2,216 (47) | 1,816 (49) | 58 (44) | 342 (40) | |||
|
| |||||||
| Age ≥ 18 mos at diagnosis | 2,546 (54) | 1,892 (51) | 83 (62) | 571 (68) | <0.0001 | 0.0112 | 0.2392 |
| Age < 18 mos at diagnosis | 2,126 (46) | 1,802 (49) | 50 (38) | 274 (32) | |||
|
| |||||||
| INSS Stage 4 | 1,927 (41) | 1,184 (32) | 84 (63) | 659 (78) | <0.0001 | <0.0001 | 0.0002 |
| All other stages | 2,741 (59) | 2,506 (68) | 49 (37) | 186 (22) | |||
|
| |||||||
| High Risk | 1,839 (40) | 955 (27) | 70 (54) | 814 (97) | <0.0001 | <0.0001 | <0.0001 |
| Low/Intermediate Risk | 2,710 (60) | 2,627 (73) | 59 (46) | 24 (3)** | |||
|
| |||||||
| Adrenal primary | 1,534 (34) | 1,084 (30) | 58 (45) | 392 (48) | <0.0001 | 0.0003 | 0.5492 |
| Other primary sites | 2,993 (66) | 2,501 (70) | 70 (55) | 422 (52) | |||
|
| |||||||
| Thoracic primary | 881 (19) | 839 (23) | 15 (12) | 27 (3) | <0.0001 | 0.0020 | <0.0001 |
| Other primary sites | 3,646 (81) | 2,746 (77) | 113 (88) | 787 (97) | |||
|
| |||||||
| High LDH (≥662.5 U/L) | 668 (50) | 391 (39) | 23 (46) | 254 (90) | <0.0001 | 0.3187 | <0.0001 |
| Normal LDH (<662.5 U/L) | 669 (50) | 613 (61) | 27 (54) | 29 (10) | |||
|
| |||||||
| High Ferritin (≥115 ng/mL) | 422 (50) | 273 (43) | 19 (54) | 130 (77) | <0.0001 | 0.1789 | 0.0049 |
| Normal Ferritin (<115 ng/mL) | 420 (50) | 366 (57) | 16 (46) | 38 (23) | |||
|
| |||||||
| Biological Features | |||||||
|
| |||||||
| Diploid | 1,500 (36) | 1,018 (31) | 33 (28) | 449 (61) | <0.0001 | 0.5158 | <0.0001 |
| Hyperdiploid | 2,667 (64) | 2,290 (69) | 85 (72) | 292 (39) | |||
|
| |||||||
| LOH/Aberration at 1p | 499 (21) | 194 (10) | 25 (40) | 280 (71) | <0.0001 | <0.0001 | <0.0001 |
| No 1p LOH/Aberration | 1,831 (79) | 1,680 (90) | 37 (60) | 114 (29) | |||
|
| |||||||
| Aberration at 11q | 358 (15) | 306 (16) | 23 (37) | 29 (7) | 0.0001 | <0.0001 | <0.0001 |
| No 11q Aberration | 1,969 (85) | 1,564 (84) | 39 (63) | 366 (93) | |||
|
| |||||||
| Presence of LOH at 1p and/or 11q | 769 (33) | 445 (24) | 38 (61) | 286 (73) | <0.0001 | <0.0001 | 0.0682 |
| No LOH at 1p or 11q | 1,559 (67) | 1,427 (76) | 24 (39) | 108 (27) | |||
|
| |||||||
| Unfavorable Histology | 1,959 (45) | 1,193 (35) | 78 (61) | 688 (91) | <0.0001 | <0.0001 | <0.0001 |
| Favorable Histology | 2,359 (55) | 2,246 (65) | 49 (39) | 64 (9) | |||
|
| |||||||
| High MKI | 712 (19) | 225 (7) | 19 (15) | 468 (68) | <0.0001 | 0.0016 | <0.0001 |
| Low/Intermediate MKI | 3,103 (82) | 2,779 (93) | 106 (85) | 218 (32) | |||
|
| |||||||
| Undifferentiated/Poorly Differentiated | 3,633 (91) | 2,750 (89) | 124 (96) | 759 (99) | <0.0001 | 0.0120 | 0.0347 |
| Differentiating | 348 (9) | 333 (11) | 5 (4) | 10 (1) | |||
Percentages reflect percent among patients with known data for each variable. Data not available as follows: stage (n=4); risk group (n=123); primary site (n=145); LDH (n=3,335); ferritin (n=3,830); ploidy (n=505); 1p status (n=2,342); 11q status (n=2,345); histology (n=354); MKI (n=857); grade of differentiation (n=691).
23 of 24 patients had initial stage 1 disease; 1 of 24 patients with MYCN fold increase > 4, but was viewed as equivocal and thus not designated as amplified.
Clinical Features Differ According to MYCN Copy Number
Clinical features were significantly associated with MYCN copy number in the full cohort (Table 1). We observed significant trends for an increasing proportion of patients with unfavorable clinical features with increasing MYCN copy number category. Some of the most prominent differences were in the proportion of patients with INSS stage 4 disease (32% for MYCN wild-type, 63% for MYCN gain, 78% for MNA; p<0.0001) and proportion of patients with elevated LDH (39% for MYCN wild-type, 46% for MYCN gain, 90% for MNA; p<0.0001). In addition, trends in primary adrenal tumor site (30% for MYCN wild-type, 45% for MYCN gain, 48% for MNA; p<0.0001) reinforced prior findings that primary adrenal tumors are more frequently associated with increased MYCN copy number.4 When this comparison was restricted to patients with MYCN gain versus MNA, it was not statistically significant. In contrast, we observed that as MYCN copy number increased, the proportion of patients with a primary thoracic tumor decreased (23% for MYCN wild-type, 12% for MYCN gain, 3% for MNA; p<0.0001).
We next compared clinical features by MYCN copy number in clinically relevant cohorts. Among patients with high-risk, stage 4 disease, most of the tests of trend of MYCN copy number with clinical features were again significant (Table 2A; n=1,543). Among patients with non-stage 4 disease, clear ordered trends were observed: the proportion of patients with thoracic primary site decreased with increasing MYCN copy number and the proportion with high LDH increased with increasing MYCN copy number (Table 2B; n=2,741).
Table 2.
Subgroup analyses of clinical and biological features according to MYCN copy number in patients with high risk, stage 4 disease (A: n = 1,543) and in patients with non-stage 4 disease (B: n = 2,741).*
| A. | High-risk, stage 4 subgroup N (%) |
A: MYCN Wild-Type N (%) |
B: MYCN Gain (signal 2–4x) N (%) |
C: MYCN Amp (signal >4x) N (%) |
p-value (A vs. B. vs. C) | p-value for Non-Amplified Groups(A vs. B) | p-value for Non-Wild-Type Groups (B vs. C) |
|---|---|---|---|---|---|---|---|
|
| |||||||
| Male | 906 (59) | 473 (57) | 44 (67) | 389 (60) | 0.2889 | 0.1336 | 0.2802 |
| Female | 637 (41) | 354 (43) | 22 (33) | 261 (40) | |||
|
| |||||||
| Age ≥ 18 mos at diagnosis | 1,316 (85) | 798 (96) | 64 (97) | 454 (70) | <0.0001 | 1.000 | <0.0001 |
| Age < 18 mos at diagnosis | 227 (15) | 29 (4) | 2 (3) | 196 (30) | |||
|
| |||||||
| Adrenal primary | 632 (43) | 298 (38) | 24 (39) | 310 (50) | <0.0001 | 0.8304 | 0.1232 |
| Other primary sites | 838 (57) | 487 (62) | 37 (61) | 314 (50) | |||
|
| |||||||
| Thoracic primary | 91 (6) | 67 (9) | 7 (11) | 17 (3) | <0.0001 | 0.4336 | 0.0004 |
| Other primary sites | 1,379 (64) | 718 (91) | 54 (89) | 607 (97) | |||
|
| |||||||
| High LDH (≥662.5 U/L) | 369 (73) | 150 (57) | 13 (52) | 206 (95) | <0.0001 | 0.6125 | <0.0001 |
| Normal LDH (<662.5 U/L) | 134 (27) | 112 (43) | 12 (48) | 10 (5) | |||
|
| |||||||
| High Ferritin (≥115 ng/mL) | 233 (77) | 117 (73) | 12 (75) | 104 (81) | 0.1064 | 1.000 | 0.5515 |
| Normal Ferritin (<115 ng/mL) | 71 (23) | 43 (27) | 4 (25) | 24 (19) | |||
|
| |||||||
| Diploid | 741 (55) | 359 (50) | 24 (39) | 358 (62) | <0.0001 | 0.1147 | 0.0004 |
| Hyperdiploid | 613 (45) | 361 (50) | 37 (61) | 215 (38) | |||
|
| |||||||
| LOH/Aberration at 1p | 327 (44) | 83 (21) | 14 (41) | 230 (75) | <0.0001 | 0.0066 | <0.0001 |
| No 1p LOH/Aberration | 409 (56) | 314 (79) | 20 (59) | 75 (25) | |||
|
| |||||||
| Aberration at 11q | 238 (32) | 195 (49) | 16 (47) | 27 (9) | <0.0001 | 0.7961 | <0.0001 |
| No 11q Aberration | 497 (68) | 200 (51) | 18 (53) | 279 (91) | |||
|
| |||||||
| Presence of LOH at 1p and/or 11q | 495 (67) | 236 (59) | 23 (68) | 236 (77) | <0.0001 | 0.3486 | 0.2050 |
| No LOH at 1p or 11q | 241 (33) | 161 (41) | 11 (32) | 69 (23) | |||
|
| |||||||
| Unfavorable Histology | 1,307 (95) | 705 (95) | 61 (98) | 541 (95) | 0.9792 | 0.3558 | 0.3490 |
| Favorable Histology | 70 (5) | 39 (5) | 1 (2) | 30 (6) | |||
|
| |||||||
| High MKI | 499 (41) | 127 (19) | 15 (25) | 357 (70) | <0.0001 | 0.2832 | <0.0001 |
| Low/Intermediate MKI | 732 (59) | 533 (81) | 45 (75) | 154 (30) | |||
|
| |||||||
| Undifferentiated/Poorly Differentiated | 1,380 (98) | 727 (97) | 63 (100) | 590 (99) | 0.0005 | 0.2473 | 1.000 |
| Differentiating | 26 (2) | 23 (3) | 0 (0) | 3 (1) | |||
| B. | Non-stage 4 subgroup N (%) |
A: MYCN Wild-Type N (%) |
B: MYCN Gain (signal 2–4x) N (%) |
C: MYCN Amp (>4x) N (%) |
p-value(A vs. B vs. C) | p-value for Non-Amplified Groups(A vs. B) | p-value for Non-Wild-Type Groups (B vs. C) |
|---|---|---|---|---|---|---|---|
|
| |||||||
| Male | 1,371 (50) | 1,239 (49) | 22 (45) | 110 (59) | 0.0199 | 0.5287 | 0.0739 |
| Female | 1,370 (50) | 1,267 (51) | 27 (55) | 76 (41) | |||
|
| |||||||
| Age ≥ 18 mos at diagnosis | 1,212 (44) | 1,082 (43) | 18 (37) | 112 (60) | <0.0001 | 0.3671 | 0.0033 |
| Age < 18 mos at diagnosis | 1,529 (56) | 1,424 (57) | 31 (63) | 74 (40) | |||
|
| |||||||
| High Risk | 296 (11) | 128 (5) | 4 (8) | 164 (89) | <0.0001 | 0.3217 | <0.0001 |
| Low/Intermediate Risk | 2,364 (89) | 2,299 (95) | 44 (92) | 21 (11) | |||
|
| |||||||
| Adrenal primary | 762 (28) | 663 (27) | 22 (45) | 77 (43) | <0.0001 | 0.0053 | 0.7676 |
| Other primary sites | 1,926 (72) | 1,795 (73) | 27 (55) | 104 (57) | |||
|
| |||||||
| Thoracic primary | 720 (27) | 705 (29) | 6 (12) | 9 (5) | <0.0001 | 0.0115 | 0.0674 |
| Other primary sites | 1,968 (73) | 1,753 (71) | 43 (88) | 172 (95) | |||
|
| |||||||
| High LDH (≥662.5 U/L) | 241 (34) | 189 (30) | 8 (50) | 44 (70) | <0.0001 | 0.0835 | 0.1351 |
| Normal LDH (<662.5 U/L) | 471 (66) | 444 (70) | 8 (50) | 19 (30) | |||
|
| |||||||
| High Ferritin (≥115 ng/mL) | 148 (32) | 121 (30) | 4 (33) | 23 (62) | <0.0001 | 0.7550 | 0.1037 |
| Normal Ferritin (<115 ng/mL) | 311 (68) | 289 (70) | 8 (67) | 14 (38) | |||
|
| |||||||
| Diploid | 699 (28) | 605 (27) | 6 (14) | 88 (54) | <0.0001 | 0.0526 | <0.0001 |
| Hyperdiploid | 1,779 (72) | 1,666 (73) | 38 (86) | 75 (46) | |||
|
| |||||||
| LOH/Aberration at 1p | 132 (10) | 78 (6) | 6 (29) | 48 (56) | <0.0001 | <0.0001 | 0.0220 |
| No 1p LOH/Aberration | 1,240 (90) | 1,188 (94) | 15 (71) | 37 (44) | |||
|
| |||||||
| Aberration at 11q | 83 (6) | 76 (6) | 5 (24) | 2 (2) | 0.5555 | 0.0009 | 0.0032 |
| No 11q Aberration | 1,287 (94) | 1,188 (94) | 16 (76) | 83 (98) | |||
|
| |||||||
| Presence of LOH at 1p and/or 11q | 201 (15) | 144 (11) | 9 (43) | 48 (56) | <0.0001 | <0.0001 | 0.2625 |
| No LOH at 1p or 11q | 1,168 (85) | 1,119 (89) | 12 (57) | 37 (44) | |||
|
| |||||||
| Unfavorable Histology | 618 (24) | 461 (19) | 16 (33) | 141 (82) | <0.0001 | 0.0170 | <0.0001 |
| Favorable Histology | 1,970 (76) | 1,906 (81) | 32 (67) | 32 (18) | |||
|
| |||||||
| High MKI | 191 (9) | 80 (4) | 4 (8) | 107 (64) | <0.0001 | 0.1284 | <0.0001 |
| Low/Intermediate MKI | 2,040 (91) | 1,936 (96) | 44 (92) | 60 (36) | |||
|
| |||||||
| Undifferentiated/Poorly Differentiated | 1,887 (86) | 1,683 (85) | 43 (90) | 161 (96) | <0.0001 | 0.3590 | 0.0955 |
| Differentiating | 314 (14) | 302 (15) | 5 (10) | 7 (4) | |||
Percentages reflect percent among patients with known data for each variable. For high-risk, stage 4 cohort, data not available as follows: primary site (n=73); LDH (n=1,040); ferritin (n=1,239); ploidy (n=189); 1p status (n=807); 11q status (n=808); histology (n=166); MKI (n=312); grade of differentiation (n=137). For non-stage 4 cohort, data not available as follows: risk group (n=81); primary site (n=53); LDH (n=2,029); ferritin (n=2,282); ploidy (n=263); 1p status (n=1,369); 11q status (n=1,371); histology (n=153); MKI (n=510); grade of differentiation (n=540).
Biological Features Differ According to MYCN Copy Number
A number of strong associations between unfavorable biological features and higher MYCN copy number were detected in the full cohort (Table 1), including: 1p LOH; unfavorable histology; high MKI; and un-/poorly differentiated grade. However, the highest rate of aberration at 11q was observed in patients with MYCN gain (37%) compared to the other groups (16% for wild-type and 7% for MNA tumors). Comparing MYCN gain and MNA, some of the most prominent differences in biological features were for 1p LOH (40% in gain vs. 71% in MNA) and for high MKI (15% in gain vs. 68% for MNA).
In our subgroup analyses of high-risk, stage 4 disease (Table 2A) and of non-stage 4 disease (Table 2B), we observed similar findings for 1p LOH and MKI as were observed in the full cohort. However, the association with histology observed in the full cohort was only seen among patients with non-stage 4 disease, perhaps due to small numbers of patients with high-risk, stage 4 disease with favorable histology. Interestingly, a similar pattern for aberration at 11q was observed among patients with non-stage 4 disease as was seen in the full cohort, with patients with MYCN gain having the highest proportion of tumors with 11q aberration (24%). In contrast, among patients with high-risk stage 4 disease, patients with wild-type and MYCN gain tumors had similar rates of 11q aberration (49% and 47%, respectively), with patients with MNA again having very low rates of 11q aberration (9%).
Clinical Outcomes Differ According to MYCN Copy Number
In the full cohort, EFS differed significantly according to MYCN copy number category (Figure 1A). Specifically, the 5-year EFS for patients with wild-type disease was 76.4% (95% CI 73.8–79.0%) compared to 57.4% (95% CI 39.6–75.2%) for patients with MYCN gain and 48.1% (95% CI 42.3–54%) for patients with MNA (p<0.0001). We also investigated specific subgroups of patients (high risk, stage 4; non-stage 4; and high-risk) and again observed significant differences in EFS according to MYCN copy number (Figure 1B, 1C, and 1D; p-values adjusted for non-proportional hazards; see Supplemental Table 3 for comparisons of patients with MYCN gain vs. MNA and for patients with MYCN gain vs. wild-type).
Figure 1.
Kaplan-Meier estimates of event-free survival for the full cohort (A), high-risk stage 4 patients (B), high-risk patients (C) and non-stage 4 patients (D).
*Global p-value for effect of MYCN copy number from univariate Cox model adjusted for non-proportional hazards.
OS likewise differed significantly according to MYCN copy number category (Figure 2A–E). In the full cohort, the 5-year OS for patients with wild-type disease was 86.1% (95% CI 84.0–88.2%) compared to 66.3% (95% CI 49.5–83.2%) for patients with MYCN gain and 55.2% (95% CI 49.4–60.9%) for patients with MNA (p<0.0001, adjusted for non-proportional hazards; Figure 2A). We observed that subgroups with non-stage 4 disease or non-high-risk disease were at increased risk of death in the presence of MYCN gain, with OS significantly lower than patients with MYCN wild-type tumors in both subgroups (unadjusted p < 0.0001 and 0.0017, respectively; Figure 2D–E and Supplemental Table 3). Deaths among patients with MYCN gain in the non-stage 4 subgroup or in the non-high-risk subgroup were exclusively seen in the first year after diagnosis (Figure 2D–E). In contrast, among patients with high-risk disease, OS was not significantly different between patients with MYCN gain vs. MYCN wild-type tumors (Supplemental Table 3).
Figure 2.
Kaplan-Meier estimates of overall survival for the full cohort (A), high-risk stage 4 patients (B), high-risk patients (C), non-stage 4 patients (D), and non-high-risk patients (E).
*Global p-value for effect of MYCN copy number from univariate Cox model adjusted for non-proportional hazards.
In Cox models of EFS and OS, after controlling for age, INSS stage, ploidy, MKI, and grade of differentiation, the outcome for patients with MYCN gain was no longer statistically significantly lower compared to patients with MYCN wild-type tumors (Table 3). The presence of MNA remained prognostic of lower EFS and OS. Among high-risk patients (subgroups of high-risk patients or stage 4, high-risk patients), we observed similar findings. Among patients with more favorable features (non-stage 4 subgroup and non-high-risk subgroup), we demonstrated that patients with MYCN gain had inferior OS even after controlling for other risk factors (adjusted p = 0.0262 and 0.0331, respectively).
Table 3.
Prognostic impact of MYCN copy number category on multivariable analyses of EFS and OS.&
| MYCN copy number category | N | Multivariable Hazard Ratio* (95% CI) | p-value | Gain vs. Amp. Contrast p-value (Gain is reference) |
|---|---|---|---|---|
| Event-Free Survival (Full Cohort) | ||||
| MYCN wild-type | 3,694 | Reference | -- | -- |
| MYCN gain | 133 | 1.192 (0.812, 1.749) | 0.3691 | 0.5097 |
| MYCN amp | 845 | 1.364 (1.128, 1.650) | 0.0014 | |
| Overall Survival (Full Cohort) | ||||
| MYCN wild-type | 3,694 | Reference | -- | -- |
| MYCN gain | 133 | 1.394 (0.871, 2.229) | 0.1662 | 0.1874 |
| MYCN amp | 845 | 3.168 (2.270, 4.421) | [<0.0001] | |
| Event-Free Survival (High-Risk, Stage 4 Patients) | ||||
| MYCN wild-type | 827 | Reference | -- | -- |
| MYCN gain | 66 | 1.117 (0.722, 1.728) | 0.6179 | 0.7520 |
| MYCN amp | 650 | 2.065 (1.468, 2.903) | [<0.0001] | |
| Overall Survival (High-Risk, Stage 4 Patients) | ||||
| MYCN wild-type | 827 | Reference | -- | -- |
| MYCN gain | 66 | 1.068 (0.630, 1.810) | 0.8083 | 0.0208 |
| MYCN amp | 650 | 3.688 (2.468, 5.512) | [<0.0001] | |
| Event-Free Survival (High-Risk Patients) | ||||
| MYCN wild-type | 955 | Reference | -- | -- |
| MYCN gain | 70 | 1.087 (0.704, 1.680) | 0.7055 | 0.6144 |
| MYCN amp | 814 | 2.188 (1.578, 3.033) | [<0.0001] | |
| Overall Survival (High-Risk Patients) | ||||
| MYCN wild-type | 955 | Reference | -- | |
| MYCN gain | 70 | 1.049 (0.620, 1.777) | 0.8574 | [0.0122] |
| MYCN amp | 814 | 3.863 (2.628, 5.677) | [<0.0001] | |
| Event-Free Survival (Non-Stage 4 Patients) | ||||
| MYCN wild-type | 2,506 | Reference | -- | -- |
| MYCN gain | 45 | 14.390 (3.447, 60.071) | [0.0003] | {0.0378} |
| MYCN amp | 185 | 1.472 (0.895, 2.421) | 0.1274 | |
| Overall Survival (Non-Stage 4 Patients) | ||||
| MYCN wild-type | 2,506 | Reference | -- | -- |
| MYCN gain | 45 | 3.804 (1.171, 12.358) | 0.0262 | 0.9407 |
| MYCN amp | 185 | 3.993 (1.936, 8.234) | 0.0002 | |
| Event-Free Survival (Non High-Risk Patients) | ||||
| MYCN wild-type | 2,627 | Reference | -- | -- |
| MYCN gain | 59 | 1.335 (0.591, 3.013) | 0.4870 | -- |
| Overall Survival (Non High-Risk Patients) | ||||
| MYCN wild-type | 2,627 | Reference | -- | -- |
| MYCN gain | 59 | 3.068 (1.094, 8.606) | 0.0331 | -- |
Non-proportional hazards are indicated by bracketed [] and braced {} p-values. Brackets indicate decreasing hazard over time, braces indicate increasing hazard over time, with the corresponding hazard ratios presented in the table representing the initial estimated hazard ratio at the beginning of observation, i.e. at diagnosis.
Adjusted for age, INSS stage (full cohort and high-risk subgroup only), ploidy, MKI and grade of differentiation.
In the full cohort and in the non-stage 4 cohort, we constructed separate Cox models of EFS and OS controlling for 11q status to determine if the aforementioned associations between MYCN gain and prognosis were potentially confounded by the association between MYCN gain and 11q aberration. In both cohorts, the presence of MYCN gain remained prognostic independent of 11q status (Supplemental Table 4).
To provide context for these observed differences, we investigated potential differences in induction response rates by copy number category for high-risk patients treated on COG protocols ANBL0532 and ANBL12P1 (Table 4). Data were available for 687 patients (364 with MYCN wild-type tumors; 24 with MYCN gain tumors; and 299 with MNA). Of these, data represent end-induction response in 665 patients and mid-induction response in 22 patients who withdrew prior to end-induction. We found the highest response rate (partial response or better) among those with MYCN amplification (85.0%) and lowest response rate among those with MYCN gain (62.5% in MYCN gain; p=0.0004). Likewise, patients with MYCN amplified disease had the highest complete response rate (25.1%), patients with MYCN gain had the lowest complete response rate (0%), and patients with wild-type tumors had an intermediate complete response rate (12.4%; p<0.0001).
Table 4.
End-induction response to chemotherapy according to MYCN copy number category for patients with high-risk neuroblastoma treated on ANBL0532 or ANBL12P1 (n = 687).
| Variable | All Patients N (%) N = 687 |
MYCN Wild-Type N (%) N = 364 |
MYCN Gain (signal 2–4x) N (%) N = 24 |
MYCN Amp (signal >4x) N (%) N = 299 |
p-value |
|---|---|---|---|---|---|
| Response Category | |||||
| Complete Response | 120 (17.5) | 45 (12.4) | 0 (0.0) | 75 (25.0) | |
| Very Good Partial Response | 177 (25.8) | 76 (20.9) | 6 (25.0) | 95 (31.8) | |
| Partial Response | 239 (34.8) | 146 (40.1) | 9 (37.5) | 84 (28.1) | |
| Mixed Response | 60 (8.7) | 46 (12.6) | 1 (4.2) | 13 (4.4) | |
| No Response | 36 (5.2) | 29 (8.0) | 3 (12.5) | 4 (1.3) | |
| Progressive Disease | 55 (8.0) | 22 (6.0) | 5 (20.8) | 28 (9.4) | |
| Responder vs. Non-Responder | |||||
| Partial Response or Better | 536 (78.0) | 267 (73.4) | 15 (62.5) | 254 (85.0) | 0.0004* |
| Less than Partial Response | 151 (22.0) | 97 (26.7) | 9 (37.5) | 45 (15.1) | |
| Complete Response (CR) vs. Non-CR | |||||
| Complete Response | 120 (17.5) | 45 (12.4) | 0 (0.0) | 75 (25.1) | <0.0001* |
| Less than Complete Response | 567 (82.5) | 319 (87.6) | 24 (100.0) | 224 (74.9) | |
From a two-sided Cochran-Armitage trend test
Discussion
MNA is a known hallmark of aggressive disease in neuroblastoma. As MYCN copy number is a discrete value and amplification is defined arbitrarily at a specific signal threshold (>4x increase by the COG and by international consensus20), it is important to understand the distribution of MYCN copy number in this disease and how clinical and biological features differ across copy number groups. Knowledge of differing prognosis according to MYCN copy number may also have therapeutic implications. Our analysis demonstrates ordered associations between MYCN copy number and several clinical and biological features of neuroblastoma as well as an association of higher MYCN copy number with lower EFS and OS. Our key findings include inferior outcomes in patients with MYCN gain in subgroups with otherwise more favorable features, the high rate of 11q aberration in tumors with MYCN gain, and the extreme rarity of low-level MNA.
The negative prognostic impact of MYCN gain may enable identification of patients with non high-risk disease who require therapy modification. While EFS and OS differed significantly among MYCN copy number groups in our full cohort (Figures 1A and 2A), our multivariable and subgroup analyses indicate that the greatest impact was in the non-stage 4 and non-high-risk subgroups. We observed inferior OS for patients with MYCN gain compared to MYCN wild-type copy number in these two subgroups (Figure 2D–E). However, we note that the absolute difference in OS is relatively modest. Our findings suggest that current management strategies may not be adequate for this unique subset of patients, though the etiology for these differences is not clear from our available data. Specifically, it is possible that the presence of MYCN gain confers a survival disadvantage directly by increasing the level of MYCN protein or MYCN-target genes, though prior studies have not consistently observed an increase in MYCN RNA with increasing MYCN copy number.23,31 Alternatively, it is possible that MYCN gain is a marker for gain of 2p and therefore a surrogate for propensity for segmental chromosomal aberrations.32 The associations between both 11q (another segmental chromosomal aberration) and hyperdiploidy (in non-stage 4 patients) and MYCN gain are consistent with this latter hypothesis. Importantly, the presence of MYCN gain remained a significant predictor of EFS and OS after controlling for 11q status.
Our findings for 11q aberration patterns support prior literature findings that 11q aberration has an inverse association with MNA.15,19 Based upon this pattern, one might reasonably expect that tumors with MYCN gain would also have lower rates of 11q aberration. Two prior groups have reported higher rates of 11q aberration in tumors with MYCN gain22,23 and we now confirm and extend this finding. In our analysis of non-stage 4 patients, 11q aberration occurred in 24% of MYCN gain tumors but in only 6% and 2% respectively of wild-type and MNA tumors. Among patients with high-risk stage 4 disease, both wild-type and MYCN gain tumors had elevated 11q aberration rates (49% and 47%, respectively) compared to patients with MNA (9%). These findings further the association of 11q aberration with high-risk disease specifically in the absence of MNA. Recent findings provide a potential link between this inverse association between 11q aberration and MNA. Specifically, evidence suggests that dysregulation of the micro-RNA let-7 plays a central role in the pathogenesis of neuroblastoma and that either MNA or 11q loss are able to disrupt let-7.33 These findings together with our current findings related to MYCN gain suggest that a threshold level of increased MYCN copy number is required to disrupt let-7. It is possible that tumors with MYCN copy number below this threshold are under genetic pressure to acquire other features, such as 11q aberration, that also disrupt this critical micro-RNA. Thus, our findings are consistent with the evolving understanding of key biological pathways of neuroblastoma oncogenesis.
We demonstrate that the incidence of low-level MYCN amplification is exceedingly low (0.2%). The reason for this paucity of cases is not clear, but is consistent with different mechanisms leading to low-level copy number increases vs. amplification. This epidemiologic finding provides reason to suggest the consolidation of low-level and high-level MYCN amplification into one category, simply MYCN amplified. Aside from 11q aberration, our findings indicate that the presence of unfavorable clinical and biological features as well as poor prognosis follow ordered patterns according to increasing MYCN copy number category. Current FISH technology allows identification of distinct copy number values for MYCN if present in single digits; however, in most cases of MNA, distinct numerical values are difficult to reliably assign. Furthermore, as more groups adopt technologies such as single nucleotide polymorphism (SNP) assays to detect MYCN copy number, the reliable identification of lower levels of MNA may be challenging. In addition, detection of heterogeneous levels of MNA, which have been the subject of other reports,34,35 will also not be feasible.
A major strength of our study was our large cohort of 4,672 contemporary patients with available MYCN copy number data, compared to 659 patients for the next largest series that has focused on patients with MYCN gain tumors.23 Moreover, all patients had MYCN testing in a centralized lab using one methodology. As stated above, we were not able to evaluate copy number as a discrete, rather than ordinal, variable. We also note that our findings represent epidemiological associations that cannot address the underlying biology that leads to these observations. If our novel finding of lower response rates to conventional chemotherapy in patients with MYCN gain and high-risk disease are seen in future studies in patients with non high-risk disease, this may suggest one possible mechanism for the inferior outcomes associated with MYCN gain in that population. We note that the basis for this lower response rate in patients with MYCN gain is as yet unknown and merits further study, including potential impact of 11q aberration on chemotherapy response. The lack of data for a subset of our outcome variables is another limitation of our work. Finally, future studies should investigate heterogeneity of MYCN copy number alterations within the same tumor. This phenomenon occurs in <2% of neuroblastoma,34,35 and was not investigated as part of our study.
In summary, we have added to our overall understanding of MYCN copy number and its association with key clinical, biological and outcome variables. Our findings demonstrate the rarity of low-level MYCN amplification, trends in associations between MYCN copy number clinical and biological features, and evidence for a specific association of 11q aberration with MYCN gain. Further studies should be conducted to elucidate the biological basis for these associations. Moreover, our work suggests that the presence of MYCN gain may identify a group of patients with otherwise more favorable features who are at higher risk of early treatment failure.
Supplementary Material
Acknowledgments
Grant Support: Supported by NIH/NCI Grants U10 CA180899, U10 CA098543, U24 CA114766, U10 CA180886, and St. Baldrick’s Foundation.
The authors would like to acknowledge staff at the COG Neuroblastoma Reference Laboratory in the Biopathology Center at Nationwide Children’s Hospital for the daily efforts that made this study possible. Specifically, we acknowledge the clinical directors who review and sign out cases: Caroline Astbury; Robert Pyatt; Shalini Reshmi; and Ruthann Pfau, as well as the pathologists who review samples: Nilsa C Ramirez and Samir Kahwash.
Footnotes
Author Contributions: Conceptualization, RB, WBL, JRP, SGD; Formal Analysis, KC, AN, CVR, SGD; Investigation, JMGF, MM, HS, MMG, MDH, JRP; Resources, MSI, MMG, MDH, JRP; Data Curation, KC, JMGF, MM, AN, CVR, SGD; Writing – original draft, KC, AN, SGD; Writing – review & editing, All; Visualization, AN, CVR; Supervision, JMGF, RB, MSI, MDH, JRP, SGD.
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