Studies published in 1994 showed clonality in Langerhans cell histiocytosis (LCH) using polymorphisms of methylation-specific restriction enzyme sites on the X-chromosome regions coding for the human androgen receptor, DXS255, PGK, and HPRT.[1,2] The results of biopsies of lesions with single-system or multisystem disease showed a proliferation of LCH cells from a single clone. The discovery of recurring genomic alterations (primarily BRAF V600E) in LCH (see below) confirmed the clonality of LCH in children.
Pulmonary LCH in adults was initially reported to be nonclonal in approximately 75% of cases,[3] while an analysis of BRAF mutations showed that 25% to 50% of adult lung LCH patients had evidence of BRAF V600E mutations.[3,4] Another study of 26 pulmonary LCH cases found that 50% had BRAF V600E mutations and 40% had NRAS mutations.[5] Approximately the same number of mutations are polyclonal, rather than monoclonal. It has not yet been investigated whether clonality and BRAF pathway mutations are concordant in the same patients, which might suggest a reactive rather than a neoplastic condition in smoker's lung LCH and a clonal neoplasm in other types of LCH.
ENLARGEFigure 11. Courtesy of Rikhia Chakraborty, Ph.D. Permission to reuse the figure in any form must be obtained directly from Dr. Chakraborty.
The genomic basis of LCH was advanced by a 2010 report of an activating mutation of the BRAF oncogene (V600E) that was detected in 35 of 61 cases (57%).[6] Multiple subsequent reports have confirmed the presence of BRAF V600E mutations in 50% or more of LCH cases in children.[7-9] Other BRAF mutations that result in signal activation have been described.[8,10] ARAF mutations are infrequent in LCH but, when present, can also lead to RAS-MAPK pathway activation.[11]
The RAS-MAPK signaling pathway (refer to Figure 11) transmits signals from a cell surface receptor (e.g., a growth factor) through the RAS pathway (via one of the RAF proteins [A, B, or C]) to phosphorylate MEK and then the extracellular signal-regulated kinase (ERK), which leads to nuclear signals affecting cell cycle and transcription regulation. The V600E mutation of BRAF leads to continuous phosphorylation, and thus activation, of MEK and ERK without the need for an external signal. Activation of ERK occurs by phosphorylation, and phosphorylated ERK can be detected in virtually all LCH lesions.[6,12]
Because RAS-MAPK pathway activation can be detected in all LCH cases, but not all cases have BRAF mutations, the presence of genomic alterations in other components of the pathway was suspected. The following genomic alterations were identified:
Whole-exome sequencing of BRAF-mutated versus BRAF–wild-type LCH biopsy tissue samples revealed that 7 of 21 BRAF–wild-type specimens had MAP2K1 mutations, while no BRAF-mutated specimens had MAP2K1 mutations.[12] The mutations in MAP2K1(which codes for MEK) were activating, as indicated by their induction of ERK phosphorylation.[12]
Another study showed MAP2K1 mutations exclusively in 11 of 22 BRAF–wild-type cases.[13]
Finally, in-frame BRAF deletions and in-frame FAM73A-BRAF fusions have occurred in the group of BRAF V600E and MAP2K1 mutation–negative cases.[14]
Studies support the universal activation of ERK in LCH, with activation in most cases being explained by BRAF and MAP2K1 alterations.[6,12,14] Altogether, these mutations in the MAP kinase pathway account for nearly 90% of the causes of the universal activation of ERK in LCH.[6,12,14]
The presence of the BRAF V600E mutation in blood and bone marrow was studied in a series of 100 patients, 65% of whom tested positive for the BRAF V600E mutation by a sensitive quantitative polymerase chain reaction technique.[7] Circulating cells with the BRAF V600E mutation could be detected in all high-risk patients and in a subset of low-risk multisystem patients. The presence of circulating cells with the mutation conferred a twofold increased risk of relapse. In a similar study that included 48 patients with BRAFV600E–mutated LCH, the BRAF V600E allele was detected in circulating cell-free DNA in 100% of patients with risk-organ–positive multisystem LCH, 42% of patients with risk-organ–negative LCH, and 14% of patients with single-system LCH.[15]
The myeloid dendritic cell origin of LCH was confirmed by finding CD34-positive stem cells with the mutation in the bone marrow of high-risk patients. In those with low-risk disease, the mutation was found in more mature myeloid dendritic cells, suggesting that the stage of cell development at which the somatic mutation occurs is critical in defining the extent of disease in LCH. LCH is now considered a myeloid neoplasm.
Clinical implications
Clinical implications of the described genomic findings include the following:
LCH joins a group of other pediatric entities with activating BRAF mutations, including select nonmalignant conditions (e.g., benign nevi) [16] and low-grade malignancies (e.g., pilocytic astrocytoma).[17,18] All of these conditions have a generally indolent course, with spontaneous resolution occurring in some cases. This distinctive clinical course may be a manifestation of oncogene-induced senescence.[16,19]
BRAF V600E mutations can be targeted by BRAF inhibitors (e.g., vemurafenib and dabrafenib) or by the combination of BRAF inhibitors plus MEK inhibitors (e.g., dabrafenib/trametinib and vemurafenib/cobimetinib). These agents and combinations are approved for adults with melanoma. Treatment of adults with combinations of a BRAF inhibitor and a MEK inhibitor showed significantly improved progression-free survival outcome compared with treatment using a BRAF inhibitor alone.[20,21] The most serious side effect of BRAF inhibitor therapies is the induction of cutaneous squamous cell carcinomas,[20,21] with the incidence of these second cancers increasing with age;[22] this effect can be reduced by concurrent treatment with both BRAF and MEK inhibitors.[20,21]
Case reports have described activity of BRAF inhibitors against LCH in adult patients [23-27] and pediatric patients,[28] but there are insufficient data to assess the role of these agents in the treatment of children with LCH.
With additional research, the observation of BRAF V600E (or potentially mutated MAP2K1) in circulating cells or cell-free DNA may become a useful diagnostic tool to define high-risk versus low-risk disease.[7] Additionally, for patients who have a somatic mutation, persistence of circulating cells with the mutation may be useful as a marker of residual disease.[7]
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Larkin J, Ascierto PA, Dréno B, et al.: Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N Engl J Med 371 (20): 1867-76, 2014. [PUBMED Abstract]
Long GV, Stroyakovskiy D, Gogas H, et al.: Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase 3 randomised controlled trial. Lancet 386 (9992): 444-51, 2015. [PUBMED Abstract]
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Neuroblastoma
Children with neuroblastoma can be subdivided into subsets with different predicted risks of relapse on the basis of clinical factors and biological markers at the time of diagnosis. Patients classified as low-risk or intermediate-risk have a favorable prognosis, with survival rates exceeding 95%. In contrast, the prognosis is more guarded for patients with high-risk neuroblastoma, with less than a 50% long-term survival rate.
Low-risk and intermediate-risk neuroblastoma usually occur in children younger than 18 months. These tumors commonly have gains of whole chromosomes and are hyperdiploid when examined by flow cytometry.[1,2]
In contrast, high-risk neuroblastoma generally occurs in children older than 18 months, is often metastatic to bone, and segmental chromosome abnormalities (gains or losses) and/or MYCN gene amplification is usually detected in these tumors. They are near diploid or near tetraploid by flow cytometric measurement.[1-7] High-risk tumors may rarely harbor exonic mutations, (refer to the Exonic mutations in neuroblastoma section of this summary for more information), but most high-risk tumors lack such gene mutations. Compared with adult cancers, neuroblastoma tumors show a low number of mutations per genome that affect protein sequence (10–20 per genome).[8]
Key genomic characteristics of high-risk neuroblastoma that are discussed below include the following:
Segmental chromosomal aberrations.
MYCN gene amplifications.
Low rates of exonic mutations, with activating mutations in ALK being the most common recurring alteration.
Genomic alterations that promote telomere lengthening.
Segmental chromosomal aberrations
Segmental chromosomal aberrations, found most frequently in 1p, 1q, 3p, 11q, 14q, and 17p are best detected by comparative genomic hybridization and are seen in most high-risk and/or stage 4 neuroblastomas.[3-7] Among all patients with neuroblastoma, a higher number of chromosome breakpoints (i.e., a higher number of segmental chromosome aberrations) correlated with the following:[3-7][Level of evidence: 3iiD]
Advanced age at diagnosis.
Advanced stage of disease.
Higher risk of relapse.
Poorer outcome.
An international collaboration studied 556 patients with high-risk neuroblastoma and identified two types of segmental copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were found in 6% of patients and were associated with a 10-year survival rate of only 3.4%; amplifications of regions not encompassing the MYCN locus, in addition to MYCN amplification, were detected in 18% of the patients and were associated with a 10-year survival rate of 5.8%.[9]
In a study of children older than 12 months who have unresectable primary neuroblastomas without metastases, segmental chromosomal aberrations were found in most, and older children were more likely to have them and to have more of them per tumor cell. In children aged 12 to 18 months, the presence of segmental chromosomal aberrations had a significant effect on event-free survival (EFS) but not on overall survival (OS). However, in children older than 18 months, there was a significant difference in OS between children with segmental chromosomal aberrations (67%) and children without segmental chromosomal aberrations (100%), regardless of tumor histology.[7]
Segmental chromosomal aberrations are also predictive of recurrence in infants with localized unresectable or metastatic neuroblastoma without MYCN gene amplification.[1,2]
MYCN gene amplification
MYCN amplification is detected in 16% to 25% of neuroblastoma tumors.[10] Among patients with high-risk neuroblastoma, 40% to 50% of cases show MYCN amplification.[11] In all stages of disease, amplification of the MYCN gene strongly predicts a poorer prognosis, in both time to tumor progression and OS, in almost all multivariate regression analyses of prognostic factors.[1,2] Within the localized tumor MYCN-amplified cohort, patients with hyperdiploid tumors have better outcomes than do patients with diploid tumors.[12] However, patients with hyperdiploid tumors with MYCN amplification or any segmental chromosomal aberrations do relatively poorly compared with patients with hyperdiploid tumors without MYCN amplification.[3]
In a Children’s Oncology Group study of MYCN copy number in 4,672 patients with neuroblastoma, 79% had MYCN–wild-type tumors, 3% had tumors with MYCN gain (defined as a twofold to fourfold increase in signal by fluorescence in situ hybridization), and 18% had MYCN-amplified tumors. When individual clinical/biological features were examined, the percentage of patients with unfavorable features was lowest in the MYCN–wild-type category, intermediate in the MYCN-gain category, and highest in the MYCN-amplified category (P < .0001), except for the tumors with 11q aberration, for which the highest rates were in the MYCN-gain category. Patients with non–stage 4 disease and patients with non–high-risk disease and MYCN gain had a significantly increased risk of death than did patients with MYCN–wild-type tumors.[13]
Most unfavorable clinical and pathobiological features are associated, to some degree, with MYCN amplification; in a multivariable logistic regression analysis of 7,102 International Neuroblastoma Risk Group patients, pooled segmental chromosomal aberrations and gains of 17q were poor prognostic features even when not associated with MYCN amplification. However, another poor prognostic feature, segmental chromosomal aberrations at 11q, are almost entirely mutually exclusive of diffuse MYCN amplification.[14,15]
Exonic mutations in neuroblastoma
Multiple reports have documented that a minority of high-risk neuroblastomas have a low incidence of recurrently mutated genes. The most commonly mutated gene is ALK, which is mutated in approximately 10% of patients (see below). Other genes with even lower frequencies of mutations include ATRX, PTPN11, ARID1A, and ARID1B.[16-22] As shown in Figure 12, most neuroblastoma cases lack mutations in genes that are altered in a recurrent manner.
ENLARGEFigure 12. Data tracks (rows) facilitate the comparison of clinical and genomic data across cases with neuroblastoma (columns). The data sources and sequencing technology used were whole-exome sequencing (WES) from whole-genome amplification (WGA) (light purple), WES from native DNA (dark purple), Illumina WGS (green), and Complete Genomics WGS (yellow). Striped blocks indicate cases analyzed using two approaches. The clinical variables included were sex (male, blue; female, pink) and age (brown spectrum). Copy number alterations indicates ploidy measured by flow cytometry (with hyperdiploid meaning DNA index >1) and clinically relevant copy number alterations derived from sequence data. Significantly mutated genes are those with statistically significant mutation counts given the background mutation rate, gene size, and expression in neuroblastoma. Germline indicates genes with significant numbers of germline ClinVar variants or loss-of-function cancer gene variants in our cohort. DNA repair indicates genes that may be associated with an increased mutation frequency in two apparently hypermutated tumors. Predicted effects of somatic mutations are color coded according to the legend. Reprinted by permission from Macmillan Publishers Ltd: Nature GeneticsExit Disclaimer (Pugh TJ, Morozova O, Attiyeh EF, et al.: The genetic landscape of high-risk neuroblastoma. Nat Genet 45 (3): 279-84, 2013), copyright (2013).
ALK, the exonic mutation found most commonly in neuroblastoma, is a cell surface receptor tyrosine kinase, expressed at significant levels only in developing embryonic and neonatal brains. Germline mutations in ALK have been identified as the major cause of hereditary neuroblastoma. Somatically acquired ALK-activating exonic mutations are also found as oncogenic drivers in neuroblastoma.[21]
The presence of an ALK mutation correlates with significantly poorer survival in high-risk and intermediate-risk neuroblastoma patients. ALK mutations were examined in 1,596 diagnostic neuroblastoma samples.[21] ALK tyrosine kinase domain mutations occurred in 8% of samples—at three hot spots and 13 minor sites—and correlated significantly with poorer survival in patients with high-risk and intermediate-risk neuroblastoma. ALKmutations were found in 10.9% of MYCN-amplified tumors versus 7.2% of those without MYCN amplification. ALK mutations occurred at the highest frequency (11%) in patients older than 10 years.[21] The frequency of ALK aberrations was 14% in the high-risk neuroblastoma group, 6% in the intermediate-risk neuroblastoma group, and 8% in the low-risk neuroblastoma group. The high-risk group included tumors with ALK aberrations, consisting of ALK co-amplification with MYCN amplification, which may also result in ALKactivation.
There are limited data regarding the genomic evolution of exonic mutations from diagnosis to relapse for neuroblastoma. Whole-genome sequencing was applied to 23 paired diagnostic and relapsed neuroblastoma tumor samples to define somatic genetic alterations associated with relapse,[23] while a second study evaluated 16 paired diagnostic and relapsed specimens.[24] Both studies identified an increased number of mutations in the relapsed samples compared with the samples at diagnosis; this has been confirmed in a study of neuroblastoma tumor samples sent for next-generation sequencing.[25]
In the first study, an increased incidence of mutations in genes associated with RAS-MAPK signaling were found in tumors at relapse compared with tumors from the same patient at diagnosis; 15 of 23 relapse samples contained somatic mutations in genes involved in this pathway and each mutation was consistent with pathway activation.[23]
In addition, three relapse samples showed structural alterations involving MAPK pathway genes consistent with pathway activation, so aberrations in this pathway were detected in 18 of 23 relapse samples (78%). Aberrations were found in ALK (n = 10), NF1(n = 2), and one each in NRAS, KRAS, HRAS, BRAF, PTPN11, and FGFR1. Even with deep sequencing, 7 of the 18 alterations were not detectable in the primary tumor, highlighting the evolution of mutation presumably leading to relapse and the importance of genomic evaluations of tissues obtained at relapse.
In the second study, ALK mutations were not observed in either diagnostic or relapse specimens, but relapse-specific recurrent single-nucleotide variants were observed in 11 genes, including the putative CHD5 neuroblastoma tumor suppressor gene located at chromosome 1p36.[24]
In a deep-sequencing study, 276 neuroblastoma samples (comprised of all stages and from patients of all ages at diagnosis) underwent very deep (33,000X) sequencing of just two amplified ALK mutational hot spots, which revealed 4.8% clonal mutations and an additional 5% subclonal mutations, suggesting that subclonal ALK gene mutations are common.[26] Thus, deep sequencing can reveal the presence of mutations in tiny subsets of neuroblastoma tumor cells that may be able to survive during treatment and grow to constitute a relapse.
Lengthening of telomeres, the tips of chromosomes, promotes cell survival. Telomeres otherwise shorten with each cell replication, resulting eventually in the lack of a cell’s ability to replicate. Low-risk neuroblastoma tumors have little telomere lengthening activity. Aberrant genetic mechanisms for telomere lengthening have been identified in high-risk neuroblastoma tumors.[16,17,27] Thus far, the following three mechanisms, which appear to be mutually exclusive, have been described:
Chromosomal rearrangements involving a chromosomal region at 5p15.33 proximal to the TERT gene, which encodes the catalytic unit of telomerase, occur in approximately 25% of high-risk neuroblastoma cases and are mutually exclusive with MYCNamplifications and ATRX mutations.[16,17] The rearrangements induce transcriptional upregulation of TERT by juxtaposing the TERT coding sequence with strong enhancer elements.
Another mechanism promoting TERT overexpression is MYCN amplification,[28] which is associated with approximately 40% to 50% of high-risk neuroblastoma cases.
The ATRX mutation or deletion is found in 10% to 20% of high-risk neuroblastoma tumors, almost exclusively in older children,[18] and is associated with telomere lengthening by a different mechanism, termed alternative lengthening of telomeres.[18,27]
Additional biological factors associated with prognosis
MYC and MYCN expression
Immunostaining for MYC and MYCN proteins on a restricted subset of 357 undifferentiated/poorly differentiated neuroblastoma tumors demonstrated that elevated MYC/MYCN protein expression is prognostically significant.[29] Sixty-eight tumors (19%) highly expressed the MYCN protein, and 81 were MYCN amplified. Thirty-nine tumors (10.9%) expressed MYC highly and were mutually exclusive of high MYCN expression; in the MYC-expressing tumors, MYC or MYCN gene amplification was not seen. Segmental chromosomal aberrations were not examined in this study.[29]
Patients with favorable-histology tumors without high MYC/MYCN expression had favorable survival (3-year EFS, 89.7% ± 5.5%; 3-year OS, 97% ± 3.2%).
Patients with undifferentiated or poorly differentiated histology tumors without MYC/MYCN expression had a 3-year EFS rate of 63.1% (± 13.6%) and a 3-year OS rate of 83.5% (± 9.4%).
Three-year EFS rates in patients with MYCN amplification, high MYCN expression, and high MYC expression were 48.1% (± 11.5%), 46.2% (± 12%), and 43.4% (± 23.1%), respectively, and OS rates were 65.8% (± 11.1%), 63.2% (± 12.1%), and 63.5% (± 19.2%), respectively.
Additionally, when high expression of MYC and MYCN proteins underwent multivariate analysis with other prognostic factors, including MYC/MYCN gene amplification, high MYC and MYCN protein expression was independent of other prognostic markers.
Neurotrophin receptor kinases
Expression of neurotrophin receptor kinases and their ligands vary between high-risk and low-risk tumors. TrkA is found on low-risk tumors, and absence of its ligand NGF is postulated to lead to spontaneous tumor regression. In contrast, TrkB is found in high-risk tumors that also express its ligand, BDNF, which promotes neuroblastoma cell growth and survival.[30]
Immune system inhibition
Anti-GD2 antibodies, along with modulation of the immune system to enhance the antibody's antineuroblastoma activity, are often used to help treat neuroblastoma. The clinical effectiveness of one such antibody led to U.S. Food and Drug Administration approval of dinutuximab. The patient response to immunotherapy may, in part, be caused by variation in immune function among patients. One anti-GD2 antibody, termed 3F8, used for treating neuroblastoma exclusively at one institution, utilizes natural killer cells to kill the neuroblastoma cells. However, the natural killer cells can be inhibited by the interaction of HLA antigens and killer immunoglobulin receptor (KIR) subtypes.[31,32] This finding was confirmed and expanded by an analysis of outcomes for patients treated on the national randomized COG-ANBL0032 (NCT00026312) study with the anti-GD2 antibody dinutuximab combined with granulocyte-macrophage colony-stimulating factor and interleukin-2. The study found that certain KIR/KIR-ligand genotypes were associated with better outcomes for patients who were treated with immunotherapy.[33][Level of evidence: 1A] The presence of inhibitory KIR/KIR ligands was associated with a decreased effect of immunotherapy. Thus, the patient's immune system genes help determine response to immunotherapy for neuroblastoma. Additional studies are needed to determine whether this immune system genotyping can guide patient selection for certain immunotherapies.
(Refer to the PDQ summary on Neuroblastoma Treatment for information about the treatment of neuroblastoma.)
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Bellini A, Bernard V, Leroy Q, et al.: Deep Sequencing Reveals Occurrence of Subclonal ALK Mutations in Neuroblastoma at Diagnosis. Clin Cancer Res 21 (21): 4913-21, 2015. [PUBMED Abstract]
Kurihara S, Hiyama E, Onitake Y, et al.: Clinical features of ATRX or DAXX mutated neuroblastoma. J Pediatr Surg 49 (12): 1835-8, 2014. [PUBMED Abstract]
Mac SM, D'Cunha CA, Farnham PJ: Direct recruitment of N-myc to target gene promoters. Mol Carcinog 29 (2): 76-86, 2000. [PUBMED Abstract]
Wang LL, Teshiba R, Ikegaki N, et al.: Augmented expression of MYC and/or MYCN protein defines highly aggressive MYC-driven neuroblastoma: a Children's Oncology Group study. Br J Cancer 113 (1): 57-63, 2015. [PUBMED Abstract]
Maris JM, Matthay KK: Molecular biology of neuroblastoma. J Clin Oncol 17 (7): 2264-79, 1999. [PUBMED Abstract]
Forlenza CJ, Boudreau JE, Zheng J, et al.: KIR3DL1 Allelic Polymorphism and HLA-B Epitopes Modulate Response to Anti-GD2 Monoclonal Antibody in Patients With Neuroblastoma. J Clin Oncol 34 (21): 2443-51, 2016. [PUBMED Abstract]
Venstrom JM, Zheng J, Noor N, et al.: KIR and HLA genotypes are associated with disease progression and survival following autologous hematopoietic stem cell transplantation for high-risk neuroblastoma. Clin Cancer Res 15 (23): 7330-4, 2009. [PUBMED Abstract]
Erbe AK, Wang W, Carmichael L, et al.: Neuroblastoma Patients' KIR and KIR-Ligand Genotypes Influence Clinical Outcome for Dinutuximab-based Immunotherapy: A Report from the Children's Oncology Group. Clin Cancer Res 24 (1): 189-196, 2018. [PUBMED Abstract]
Retinoblastoma
Retinoblastoma is a tumor that occurs in heritable (25%–30%) and nonheritable (70%–75%) forms. Heritable disease is defined by the presence of a germline mutation of the RB1gene. This germline mutation may have been inherited from an affected progenitor (25% of cases) or may have occurred in a germ cell before conception or in utero during early embryogenesis in patients with sporadic disease (75% of cases). The presence of positive family history or bilateral or multifocal disease is suggestive of heritable disease.
Heritable retinoblastoma may manifest as unilateral or bilateral disease. The penetrance of the RB1 mutation (laterality, age at diagnosis, and number of tumors) is probably dependent on concurrent genetic modifiers such as MDM2 and MDM4 polymorphisms.[1,2] All children with bilateral disease and approximately 15% of patients with unilateral disease are presumed to have the heritable form, even though only 25% have an affected parent.
In heritable retinoblastoma, tumors tend to be diagnosed at a younger age than in the nonheritable form of the disease. Unilateral retinoblastoma in children younger than 1 year raises concern for heritable disease, whereas older children with a unilateral tumor are more likely to have the nonheritable form of the disease.[3]
The genomic landscape of retinoblastoma is driven by alterations in RB1 that lead to biallelic inactivation.[4,5] A rare cause of RB1 inactivation is chromothripsis, which may be difficult to detect by conventional methods.[6] Other recurring genomic changes that occur in a small minority of tumors include BCOR mutation/deletion, MYCN amplification, and OTX2 amplification.[4-6] A study of 1,068 unilateral nonfamilial retinoblastoma tumors reported that a small percentage of cases (approximately 3%) lacked evidence of RB1 loss. Approximately one-half of these cases with no evidence of RB1 loss (representing approximately 1.5% of all unilateral nonfamilial retinoblastoma) showed MYCNamplification.[5] The functional status of the retinoblastoma protein (pRb) is inferred to be inactive in retinoblastoma with MYCN amplification. This suggests that inactivation of RB1by mutation or inactive pRb is a requirement for the development of retinoblastoma, independent of MYCN amplification.[7]
(Refer to the PDQ summary on Retinoblastoma Treatment for information about the treatment of retinoblastoma.)
References
Castéra L, Sabbagh A, Dehainault C, et al.: MDM2 as a modifier gene in retinoblastoma. J Natl Cancer Inst 102 (23): 1805-8, 2010. [PUBMED Abstract]
de Oliveira Reis AH, de Carvalho IN, de Sousa Damasceno PB, et al.: Influence of MDM2 and MDM4 on development and survival in hereditary retinoblastoma. Pediatr Blood Cancer 59 (1): 39-43, 2012. [PUBMED Abstract]
Zajaczek S, Jakubowska A, Kurzawski G, et al.: Age at diagnosis to discriminate those patients for whom constitutional DNA sequencing is appropriate in sporadic unilateral retinoblastoma. Eur J Cancer 34 (12): 1919-21, 1998. [PUBMED Abstract]
Zhang J, Benavente CA, McEvoy J, et al.: A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature 481 (7381): 329-34, 2012. [PUBMED Abstract]
Rushlow DE, Mol BM, Kennett JY, et al.: Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies. Lancet Oncol 14 (4): 327-34, 2013. [PUBMED Abstract]
McEvoy J, Nagahawatte P, Finkelstein D, et al.: RB1 gene inactivation by chromothripsis in human retinoblastoma. Oncotarget 5 (2): 438-50, 2014. [PUBMED Abstract]
Ewens KG, Bhatti TR, Moran KA, et al.: Phosphorylation of pRb: mechanism for RB pathway inactivation in MYCN-amplified retinoblastoma. Cancer Med 6 (3): 619-630, 2017. [PUBMED Abstract]
Kidney Tumors
Wilms Tumor
Wilms tumors, similar to other pediatric embryonal neoplasms, typically arise after a limited number of genetic aberrations. One study showed the following:[1]
Wilms tumors commonly arise through more than one genetic event.
Wilms tumors show differences in gene expression and methylation patterns with different genetic aberrations.
Wilms tumors have a large number of candidate driver genes, most of which are mutated in less than 5% of Wilms tumors.
Wilms tumors have recurrent mutations in genes with common functions, with most involved in either early renal development or epigenetic regulation (e.g., chromatin modifications, transcription elongation, and miRNA).
Approximately one-third of Wilms tumor cases involve mutations in WT1, CTNNB1, or WTX.[2,3] Another subset of Wilms tumor cases results from mutations in miRNA processing genes (miRNAPG), including DROSHA, DGCR8, DICER1, and XPO5.[4-7] Other genes critical for early renal development that are recurrently mutated in Wilms tumor include SIX1 and SIX2 (transcription factors that play key roles in early renal development),[4,5] EP300, CREBBP, and MYCN.[1] Of the mutations in Wilms tumors, 30% to 50% appear to converge on the process of transcriptional elongation in renal development and include the genes MLLT1, BCOR, MAP3K4, BRD7, and HDAC4.[1] Anaplastic Wilms tumor is characterized by the presence of TP53 mutations.
Elevated rates of Wilms tumor are observed in a number of genetic disorders, including WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, Beckwith-Wiedemann syndrome, hemihypertrophy, Denys-Drash syndrome, and Perlman syndrome.[8] Other genetic causes that have been observed in familial Wilms tumor cases include germline mutations in REST and CTR9.[9,10]
The genomic and genetic characteristics of Wilms tumor are summarized below.
Wilms tumor 1 gene (WT1)
The WT1 gene is located on the short arm of chromosome 11 (11p13). WT1 is a transcription factor that is required for normal genitourinary development and is important for differentiation of the renal blastema.[11] WT1 mutations are observed in 10% to 20% of cases of sporadic Wilms tumor.[2,11,12]
Wilms tumor with a WT1 mutation is characterized by the following:
Evidence of WNT pathway activation by activating mutations in the beta-catenin gene (CTNNB1) is common.[12-14]
Loss of heterozygosity (LOH) at 11p15 is commonly observed, as paternal uniparental disomy for chromosome 11 represents a common mechanism for losing the remaining normal WT1 allele.[12,15]
Nephrogenic rests are benign foci of embryonal kidney cells that abnormally persist into postnatal life. Intralobar nephrogenic rests occur in approximately 20% of Wilms tumor cases. They are observed at high rates in cases with genetic syndromes that have WT1 mutations such as WAGR and Denys-Drash syndromes.[16] Intralobar nephrogenic rests are also observed in cases with sporadic WT1 and MLLT1 mutations.[17,18]
WT1 germline mutations are uncommon (2%–4%) in nonsyndromic Wilms tumor.[19,20]
WT1 mutations and 11p15 loss of heterozygosity were associated with relapse in patients with very low-risk Wilms tumor in one study of 56 patients who did not receive chemotherapy.[21] These findings need validation but may provide biomarkers for stratifying patients in the future.
Germline WT1 mutations are more common in children with Wilms tumor and one of the following:
WAGR syndrome, Denys-Drash syndrome,[22] or Frasier syndrome.[23]
Genitourinary anomalies, including hypospadias and cryptorchidism.
Bilateral Wilms tumor.
Unilateral Wilms tumor with nephrogenic rests in the contralateral kidney.
Stromal and rhabdomyomatous differentiation.
Syndromic conditions with germline WT1 mutations include WAGR syndrome, Denys-Drash syndrome,[22] and Frasier syndrome.[23]
WAGR syndrome. Children with WAGR syndrome are at high risk (approximately 50%) of developing Wilms tumor.[24] WAGR syndrome results from deletions at chromosome 11p13 that involve a set of contiguous genes that includes the WT1 and PAX6 genes.
Inactivating mutations or deletions in the PAX6 gene lead to aniridia, while deletion of WT1 confers the increased risk of Wilms tumor. Sporadic aniridia in which WT1 is not deleted is not associated with increased risk of Wilms tumor. Accordingly, children with familial aniridia, generally occurring for many generations, and without renal abnormalities, have a normal WT1 gene and are not at an increased risk of Wilms tumor.[25,26]
Wilms tumor in children with WAGR syndrome is characterized by an excess of bilateral disease, intralobar nephrogenic rests–associated favorable-histology (FH) tumors of mixed cell type, and early age at diagnosis.[27] The mental retardation in WAGR syndrome may be secondary to deletion of other genes, including SLC1A2 or BDNF.[28]
Germline WT1 point mutations produce genetic syndromes that are characterized by nephropathy, 46XY disorder of sex development, and varying risks of Wilms tumor.[29,30]
Denys-Drash and Frasier syndromes. Denys-Drash syndrome is characterized by nephrotic syndrome caused by diffuse mesangial sclerosis, XY pseudohermaphroditism, and increased risk of Wilms tumor (>90%). Frasier syndrome is characterized by progressive nephropathy caused by focal segmental glomerulosclerosis, gonadoblastoma, and XY pseudohermaphroditism.
WT1 mutations in Denys-Drash syndrome are most often missense mutations in exons 8 and 9, which code for the DNA binding region of WT1.[22] By contrast, WT1 mutations in Frasier syndrome typically occur in intron 9 at the KTS site, and they affect an alternative splicing, thereby preventing production of the usually more abundant WT1 +KTS isoform.[31]
Studies evaluating genotype/phenotype correlations of WT1 mutations have shown that the risk of Wilms tumor is highest for truncating mutations (14 of 17 cases, 82%) and lower for missense mutations (27 of 67 cases, 42%). The risk is lowest for KTS splice site mutations (1 of 27 cases, 4%).[29,30] Bilateral Wilms tumor was more common in cases with WT1-truncating mutations (9 of 14 cases) than in cases with WT1 missense mutations (3 of 27 cases).[29,30] These genomic studies confirm previous estimates of elevated risk of Wilms tumor for children with Denys-Drash syndrome and low risk of Wilms tumor for children with Frasier syndrome.
Late effects associated with WAGR syndrome and Wilms tumor include the following:
Children with WAGR syndrome or other germline WT1 mutations are monitored throughout their lives because they are at increased risk of developing hypertension, nephropathy, and renal failure.[32]
Patients with Wilms tumor and aniridia without genitourinary abnormalities are at lower risk but are monitored for nephropathy or renal failure.[33]
Children with Wilms tumor and any genitourinary anomalies are also at increased risk of late renal failure and are monitored. Features associated with germline WT1mutations that increase the risk of developing renal failure include the following:[32]
CTNNB1 is the most commonly mutated gene in Wilms tumor, reported to occur in 15% of patients with Wilms tumor.[1,3,12,14,34] These CTNNB1 mutations result in activation of the WNT pathway, which plays a prominent role in the developing kidney.[35] CTNNB1mutations commonly occur with WT1 mutations, and most cases of Wilms tumor with WT1mutations have a concurrent CTNNB1 mutation.[12,14,34] Activation of beta-catenin in the presence of intact WT1 protein appears to be inadequate to promote tumor development because CTNNB1 mutations are rarely found in the absence of a WT1 or WTX mutation, except when associated with a MLLT1 mutation.[3,36] CTNNB1 mutations appear to be late events in Wilms tumor development because they are found in tumors but not in nephrogenic rests.[17]
Wilms tumor gene on the X chromosome (WTX)
WTX, which is also called FAM123B, is located on the X chromosome at Xq11.1. It is altered in 15% to 20% of Wilms tumor cases.[2,3,12,37,38] Germline mutations in WTX cause an X-linked sclerosing bone dysplasia, osteopathia striata congenita with cranial sclerosis (MIM300373Exit Disclaimer).[39] Despite having germline WTX mutations, individuals with osteopathia striata congenita are not predisposed to tumor development.[39] The WTX protein appears to be involved in both the degradation of beta-catenin and in the intracellular distribution of APC protein.[36,40] WTX is most commonly altered by deletions involving part or all of the WTX gene, with deleterious point mutations occurring less commonly.[2,12,37] Most Wilms tumor cases with WTX alterations have epigenetic 11p15 abnormalities.[12]
WTX alterations are equally distributed between males and females, and WTX inactivation has no apparent effect on clinical presentation or prognosis.[2]
Imprinting Cluster Regions (ICRs) on chromosome 11p15 (WT2) and Beckwith-Wiedemann syndrome
A second Wilms tumor locus, WT2, maps to an imprinted region of chromosome 11p15.5; when it is a germline mutation, it causes Beckwith-Wiedemann syndrome. About 3% of children with Wilms tumor have germline epigenetic or genetic changes at the 11p15.5 growth regulatory locus without any clinical manifestations of overgrowth. Like children with Beckwith-Wiedemann syndrome, these children have an increased incidence of bilateral Wilms tumor or familial Wilms tumor.[28]
Approximately 80% of patients with Beckwith-Wiedemann syndrome have a molecular defect of the 11p15 domain.[41] Various molecular mechanisms underlying Beckwith-Wiedemann syndrome have been identified. Some of these abnormalities are genetic (germline mutations of the maternal allele of CDKN1C, paternal uniparental isodisomy of 11p15, or duplication of part of the 11p15 domain) but are more frequently epigenetic (loss of methylation of the maternal ICR2/KvDMR1 or gain of methylation of the maternal ICR1).[28,42]
Several candidate genes at the WT2 locus comprise the two independent imprinted domains IGF2/H19 and KIP2/LIT1.[42] Loss of heterozygosity, which exclusively affects the maternal chromosome, has the effect of upregulating paternally active genes and silencing maternally active ones. A loss or switch of the imprint for genes (change in methylation status) in this region has also been frequently observed and results in the same functional aberrations.[28,41,42]
A relationship between epigenotype and phenotype has been shown in Beckwith-Wiedemann syndrome, with a different rate of cancer in Beckwith-Wiedemann syndrome according to the type of alteration of the 11p15 region.[43] The overall tumor risk in patients with Beckwith-Wiedemann syndrome has been estimated to be between 5% and 10%, with a risk between 1% (loss of imprinting at ICR2) and 30% (gain of methylation at ICR1 and paternal 11p15 isodisomy). For patients with Beckwith-Wiedemann syndrome, the risk of developing Wilms tumor is 4.1%. Development of Wilms tumor has been reported in patients with only ICR1 gain of methylation, whereas other tumors such as neuroblastoma or hepatoblastoma were reported in patients with paternal 11p15 isodisomy.[44-46] For patients with Beckwith-Wiedemann syndrome, the relative risk of developing hepatoblastoma is 2,280 times that of the general population.[47]
Loss of imprinting or gene methylation is rarely found at other loci, supporting the specificity of loss of imprinting at 11p15.5.[48] Interestingly, Wilms tumor in Asian children is not associated with either nephrogenic rests or IGF2 loss of imprinting.[49]
Approximately one-fifth of patients with Beckwith-Wiedemann syndrome who develop Wilms tumor present with bilateral disease, and metachronous bilateral disease is also observed.[25,47,50] The prevalence of Beckwith-Wiedemann syndrome is about 1% among children with Wilms tumor reported to the National Wilms Tumor Study (NWTS).[47,51]
Other genes and chromosomal alterations
Additional genes and chromosomal alterations that have been implicated in the pathogenesis and biology of Wilms tumor include the following:
1q. Gain of chromosome 1q is associated with an inferior outcome and is the single most powerful predictor of outcome. In the presence of 1q gain, neither 1p nor 16q loss is significant.[52,53] Gain of chromosome 1q is one of the most common cytogenetic abnormalities in Wilms tumor and is observed in approximately 30% of tumors.
In an analysis of FH Wilms tumor from 1,114 patients from NWTS-5 (COG-Q9401/NCT00002611), 28% of the tumors displayed 1q gain.[52]
The 8-year event-free survival (EFS) rate was 77% for patients with 1q gain and 90% for those lacking 1q gain (P < .001). Within each disease stage, 1q gain was associated with inferior EFS.
The 8-year overall survival (OS) rate was 88% for those with 1q gain and 96% for those lacking 1q gain (P < .001). OS was significantly inferior in cases with stage I disease (P < .0015) and stage IV disease (P = .011).
16q and 1p. Additional tumor-suppressor or tumor-progression genes may lie on chromosomes 16q and 1p, as evidenced by loss of heterozygosity for these regions in 17% and 11% of Wilms tumor cases, respectively.[54]
In large NWTS studies, patients with tumor-specific loss of these loci had significantly worse relapse-free survival and OS rates. Combined loss of 1p and 16q are used to select FH Wilms tumor patients for more aggressive therapy in the current Children's Oncology Group (COG) study. However, a U.K. study of more than 400 patients found no significant association between 1p deletion and poor prognosis, but a poor prognosis was associated with 16q loss of heterozygosity.[55]
An Italian study of 125 patients, using treatment quite similar to that in the COG study, found significantly worse prognosis in those with 1p deletions but not 16q deletions.[56]
These conflicting results may arise from the greater prognostic significance of 1q gain described above. Loss of heterozygosity of 16q and 1p loses significance as independent prognostic markers in the presence of 1q gain. However, in the absence of 1q gain, loss of heterozygosity of 16q and 1p retains their adverse prognostic impact.[52] The loss of heterozygosity of 16q and 1p appears to arise from complex chromosomal events that result in 1q loss of heterozygosity or 1q gain. The change in 1q appears to be the critical tumorigenic genetic event.[57]
miRNAPG. Mutations in selected miRNAPG are observed in approximately 20% of Wilms tumor cases and appear to perpetuate the progenitor state.[1,4-7] The products of these genes direct the maturation of miRNAs from the initial pri-miRNA transcripts to functional cytoplasmic miRNAs (refer to Figure 13).[58] The most commonly mutated miRNAPG is DROSHA, with a recurrent mutation (E1147K) affecting a metal-binding residue of the RNase IIIb domain, representing about 80% of DROSHA-mutated tumors. Other miRNAPG that are mutated in Wilms tumor include DGCR8, DICER1, TARBP2, DIS3L2, and XPO5. These mutations are generally mutually exclusive, and they appear to be deleterious and result in impaired expression of tumor-suppressing miRNAs. A striking sex bias was noted in mutations for DGCR8 (located on chromosome 22q11), with 38 of 43 cases (88%) arising in girls.[4,5]
Germline mutations in miRNAPG are observed for DICER1 and DIS3L2, with mutations in the former causing DICER1 syndrome and mutations in the latter causing Perlman syndrome.
DICER1 syndrome is typically caused by inherited truncating mutations in DICER1, with tumor formation following acquisition of a missense mutation in a domain of the remaining allele of DICER1 (the RNase IIIb domain) responsible for processing miRNAs derived from the 5p arms of pre-miRNAs.[59] Tumors associated with DICER1 syndrome include pleuropulmonary blastoma, cystic nephroma, ovarian sex cord–stromal tumors, multinodular goiter, and embryonal rhabdomyosarcoma.[59] Wilms tumor is an uncommon presentation of the DICER1 syndrome. In one study, three families with DICER1 syndrome included children with Wilms tumor, with two of the Wilms tumor cases showing the typical second DICER1 mutation in the RNase IIIb domain.[60] Another study identified DICER1mutations in 2 of 48 familial Wilms tumor families.[61] Large sequencing studies of Wilms tumor cohorts have also observed occasional cases with DICER1 mutations.[5,6]
SIX1 and SIX2.SIX1 and SIX2 are highly homologous transcription factors that play key roles in early renal development and are expressed in the metanephric mesenchyme, where they maintain the mesenchymal progenitor population. The frequency of SIX1mutations is 3% to 4% in Wilms tumor, and the frequency of SIX2 mutations in Wilms tumor is 1% to 3%.[4,5] Virtually all SIX1 and SIX2 mutations are in exon 1 and result in a glutamine-to-arginine mutation at position 177. Mutations in WT1, WTX, and CTNNB1 are infrequent in cases with SIX1/SIX2 or miRNAPG mutations. Conversely, SIX1/SIX2mutations and miRNAPG mutations tend to occur together.
MLLT1. Approximately 4% of Wilms tumor cases have mutations in the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation by RNA polymerase II during early development.[18] The mutant MLLT1 protein shows altered binding to acetylated histone tails. Patients with MLLT1-mutant tumors present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests, supporting a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumor.
TP53 (tumor suppressor gene). Most anaplastic Wilms tumor cases show mutations in the TP53 tumor suppressor gene.[65-67] TP53 may be useful as an unfavorable prognostic marker.[65,66]
In a study of 118 prospectively identified patients with diffuse anaplastic Wilms tumor registered on the NWTS-5 trial, 57 patients (48%) demonstrated TP53 mutations, 13 patients (11%) demonstrated TP53 segmental copy number loss without mutation, and 48 patients (41%) lacked both (wild-type TP53 [wtTP53]). All TP53 mutations were detected by sequencing alone. Patients with stage III or stage IV disease with wtTP53had a significantly lower relapse rate and mortality rate than did patients with TP53abnormalities (P = .00006 and P = .00007, respectively). There was no effect of TP53 status on patients with stage I or stage II tumors. In-depth analysis of a subset of 39 patients with diffuse anaplastic Wilms tumor showed that 7 patients (18%) were wtTP53. These tumors demonstrated gene expression evidence of p53 pathway activation. Retrospective pathology review of wtTP53 revealed no or very low volume of anaplasia in six of seven tumors. These data support the key role of TP53 loss in the development of anaplasia in Wilms tumor and support its significant clinical influence in patients who have residual anaplastic disease after surgery.[68]
FBXW7.FBXW7, a ubiquitin ligase component, is a gene that has been identified as recurrently mutated at low rates in Wilms tumor. Mutations of this gene have been associated with epithelial-type tumor histology.[69]
9q22.3 microdeletion syndrome. Patients with 9q22.3 microdeletion syndrome have an increased risk of Wilms tumor.[70,71] The chromosomal region with germline deletion includes PTCH1, the gene that is mutated in Gorlin syndrome (nevoid basal cell carcinoma syndrome associated with osteosarcoma). 9q22.3 microdeletion syndrome is characterized by the clinical findings of Gorlin syndrome, as well as developmental delay and/or intellectual disability, metopic craniosynostosis, obstructive hydrocephalus, prenatal and postnatal macrosomia, and seizures.[70] Five patients who presented with Wilms tumor in the context of a constitutional 9q22.3 microdeletion have been reported.[71-73]
MYCN.MYCN copy number gain was observed in approximately 13% of Wilms tumor cases, and it was more common in anaplastic cases (7 of 23 cases, 30%) than in nonanaplastic cases (11.2%).[74] Activating mutations at codon 44 (p.P44L) were identified in approximately 4% of Wilms tumor cases.[74] Germline copy number gain at MYCN has been reported in a bilateral Wilms tumor case, and germline MYCNduplication was also reported for a child with prenatal bilateral nephroblastomatosis and a family history of nephroblastoma.[75]
CTR9. Inactivating CTR9 germline mutations were identified in 3 of 35 familial Wilms tumor pedigrees.[10] CTR9, which is located at chromosome 11p15.3, is a key component of the polymerase-associated factor 1 complex (PAF1c), which has multiple roles in RNA polymerase II regulation and is implicated in embryonic organogenesis and maintenance of embryonic stem cell pluripotency.
REST. Inactivating germline mutations in REST (encoding RE1-silencing transcription factor) were identified in four familial Wilms tumor pedigrees.[9] REST is a transcriptional repressor that functions in cellular differentiation and embryonic development. Most REST mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. When screened for REST mutations, 9 of 519 individuals with Wilms tumor who had no history of relatives with the disease tested positive for the mutation; some had parents who also tested positive.[9] These observations indicate that REST is a Wilms tumor predisposition gene associated with approximately 2% of Wilms tumor.
Figure 14 summarizes the genomic landscape of a selected cohort of Wilms tumor patients selected because they experienced relapse despite showing FH.[18] The 75 FH Wilms tumor cases were clustered by unsupervised analysis of gene expression data, resulting in six clusters. Five of six MLLT1-mutant tumors with available gene expression data were in cluster 3, and two were accompanied by CTNNB1 mutations. This cluster also contained four tumors with a mutation or small segment deletion of WT1, all of which also had either a mutation of CTNNB1 or small segment deletion or mutation of WTX. It also contained a substantial number of tumors with retention of imprinting of 11p15 (including all MLLT1-mutant tumors). The miRNAPG-mutated cases clustered together and were mutually exclusive with both MLLT1 and with WT1/WTX/CTNNB1-mutated cases.
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