Childhood Cancer Genetics

Updated: Aug 18, 2016
  • Author: Samuel D Esparza, MD; Chief Editor: Max J Coppes, MD, PhD, MBA  more...
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An important development in cancer research over the past 2 decades has been the recognition that genetic changes drive the pathogenesis of tumors of both adulthood and childhood. These changes can be inherited and are, therefore, found in every cell, but more often, they are somatically acquired and restricted to tumor cells. In addition, these alterations affect 3 principal categories of genes, as follows: proto-oncogenes, tumor suppressor genes, and DNA repair genes. This article briefly discusses tumor suppressor genes and then focuses on the role of proto-oncogenes in childhood cancer.


Tumor Suppressor Genes

Inactivation of tumor suppressor genes, whose products normally provide negative control of cell proliferation, contributes to malignant transformation in various cell types. Knudson first proposed that two hits, or mutations, are required for the development of retinoblastoma. [1] His prediction was subsequently supported by the cloning of the retinoblastoma tumor suppressor gene (RB1) and by functional studies of the retinoblastoma protein, Rb. The first mutation of RB1 in cases of retinoblastoma can be either constitutional or somatic, whereas the second mutation is always somatic. In the inherited form of retinoblastoma, the first mutation is present in the germline; an early onset and a high frequency of bilateral disease characterize these cases. In contrast, both mutations in nonhereditary retinoblastoma are somatic.

Like Rb protein, many of the proteins encoded by tumor suppressor genes act at specific points in the cell cycle. For example, the TP53 gene, located on chromosome 17, encodes a 53-kd nuclear protein that functions as a cell cycle checkpoint. As a transcription factor whose expression is increased by DNA damage, p53 blocks cell division at the G1 phase of the cell cycle to allow DNA repair. The TP53 gene is also capable of stimulating apoptosis of cells containing damaged DNA. [2] Targeted disruption of TP53 in the mouse leads to the development of various tumors (see image below).

Tumor suppressor genes. DNA damage increases TP53 Tumor suppressor genes. DNA damage increases TP53 levels through an ATM-dependent pathway. TP53 activates the expression of genes involved in apoptosis, cell cycle regulation (p21), and MDM2. MDM2 binds to and inhibits TP53 activity. The cyclin-dependent kinase (CDK) inhibitors p21 and p16 inhibit the activity of CDKs, such as CDK4. The CDK4-cyclinD complex normally phosphorylates the retinoblastoma protein (Rb protein), leading to release of the E2F transcription factor and cell cycle progression. Activation of p21 or p16 therefore causes cell cycle arrest. The p19ARF protein, which is encoded by the same locus as p16, also leads to cell cycle arrest by inhibiting the ability of MDM2 to inactivate TP53.

Germline mutation of one TP53 allele is found in patients with Li-Fraumeni syndrome who generally inherit a mutated TP53 gene from an affected parent. Patients with Li-Fraumeni syndrome are predisposed to sarcomas, breast cancer, brain tumors, adrenocortical cell carcinoma, and acute leukemia; they have a 50% probability of cancer development by age 30 years.

Another important class of tumor suppressor genes involved in cell cycle control and in the generation of human cancers is the cyclin-dependent kinase (CDK) inhibitors. These proteins negatively regulate the cell cycle by inhibiting CDK phosphorylation of Rb protein and include p15INK4B, p16INK4A, p18INK4C, p19INK4D, p19ARF, p21CIP1, p27KIP1, and p57KIP2. Although carcinogenic roles for the INK4B, INK4C, INK4D, CIP1, KIP1, and KIP2 genes appear to be limited, INK4A is among the most commonly mutated genes in human tumors. The p16INK4A protein is a cell-cycle inhibitor that acts by inhibiting activated cyclin D:CDK4/6 complexes, which play a crucial role in the control of the cell cycle by phosphorylating Rb protein.

Direct evidence linking the INK4A locus to tumorigenesis was provided by the targeted disruption of exon 2 of INK4A in mice. Tumors that developed in mice deficient in INK4A were enhanced by the topical application of carcinogens and ultraviolet light. This locus, however, also encodes a protein from an alternative reading frame, designated p19ARF. Interpretation of INK4A knockout experiments was uncertain because targeted disruption of exon 2 of INK4A also disrupts ARF. Further genetic analysis showed that selective disruption of ARF reproduces the phenotype described for INK4A- null mice; this finding indicates that ARF is a true tumor suppressor gene.

In addition, p19ARF binds to and promotes the degradation of MDM2, the product of the murine double minute 2 gene, and this degradation leads to accumulation of TP53 and to cell cycle arrest. Therefore, both INK4A and ARF appear to be tumor suppressor genes, acting through either the Rb protein (INK4A) pathway or the TP53 (ARF) pathway in different tumor subsets.

Although many other tumor suppressor genes are involved in human cancers, the functions of their encoded proteins are not completely understood. Similarly, the reason that germline mutations in tumor suppressor genes predispose only to specific tumors is unclear. The characterization of additional tumor suppressor genes and a better understanding of how their inactivation leads to tumorigenesis should ultimately lead to improvements in the treatment of childhood cancer.

A study by Zhang et al found germline mutations in cancer-predisposing genes were identified in 8.5% (95 patients) of the children and adolescents with cancer. The study sequenced DNA from blood samples of 1120 patients younger than 20 years of age. This included whole genomes (595 patients), whole exomes (456 patients), or both (69 patients). The study also added that family history did not predict the presence of an underlying predisposition syndrome in most patients. [3, 4]


Proto-oncogene Activation

Activation of proto-oncogenes is a common theme in childhood leukemias and solid tumors. Transcription factors (proteins that bind to the regulatory sequences of target genes) compose the largest class of oncogenes identified in pediatric tumors. Oncogenic transcription factors commonly show close homology to proteins with important regulatory functions in primitive organisms; this homology indicates that the biochemical pathways leading to cell transformation are well conserved in nature.

Tumor-specific translocations can oncogenically activate transcription factors by at least 2 mechanisms. In B-progenitor acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), and certain solid tumors, translocations fuse discrete portions of 2 different genes to create chimeric transcription factors with oncogenic properties. Gene fusion is also the mechanism by which tyrosine kinases become activated in ALL. Alternatively, in T-cell and B-cell acute leukemia, transcription factor genes are dysregulated by their juxtaposition with transcriptionally active T-cell receptor (TCR) or immunoglobulin (IG) genes.


Genetics of Childhood Acute Lymphoblastic Leukemia

BCR-abl fusion gene

The first fusion gene described in acute lymphoblastic leukemia (ALL), BCR-abl, is created by the der(22) of the t(9;22)(q34;q11), which occurs in 3-5% of childhood cases. The t(9;22) is also present in almost all cases of chronic myelogenous leukemia (CML). Among those cases of CML in which the t(9;22) is not detected, the fusion gene is detected. This translocation moves the abl proto-oncogene from chromosome 9 into the BCR gene on chromosome 22. In most CML cases and in about half of adult BCR-abl –positive ALL cases, the BCR breakpoints are located in the major breakpoint cluster region. Chimeric messenger RNAs (mRNAs) transcribed from the BCR-abl fusion gene encode a fusion tyrosine kinase of approximately 210 kd (p210), or less commonly, 230kd (p230).

In most cases of childhood BCR-abl– positive ALL, the BCR breakpoints are distributed in an upstream breakpoint cluster region; in this location, the fusion gene encodes a 190-kd chimeric protein (p190). The tyrosine kinase activity of p210 and p190 is higher than that of abl. Both p210 and p190 localize to the cytoplasm and transform hematopoietic precursors in experimental systems. Their role in leukemic transformation appears to involve multiple signaling pathways.

In pediatric ALL, the t(9;22) is associated with a poor prognosis; allogeneic hematopoietic stem cell transplantation during first remission was formerly believed to be the only curative treatment. However, evidence indicates that certain subsets of these patients, including those who have low leukocyte counts at the time of diagnosis and those whose disease initially responds well to prednisone or the addition of a BCR-abl tyrosine kinase inhibitor, may have improved survival with contemporary chemotherapy.

E2A-PBX1 fusion gene

The t(1;19)(q23;p13.3), which occurs in 25% of ALL cases with a pre-B (cytoplasmic IG-positive) immunophenotype, fuses the transactivation domains of the E2A gene, which encodes a basic helix-loop-helix (bHLH) transcription factor, to PBX1, an atypical homeobox (HOX) gene. Each of the E2A proteins (E12 and E47) contains a bHLH domain that is responsible for sequence-specific DNA binding and protein dimerization. In addition, the amino terminal portion of E2A contains 2 transcriptional transactivation domains. The E2A-PBX1 chimeric protein contains these transactivation domains fused to the homeodomain of PBX1.

E2A-PBX1 binds DNA in a site-specific manner, is a strong transcriptional transactivator, and transforms NIH3T3 fibroblasts in culture. In addition, it induces T-cell lymphomas in transgenic mice and, when introduced into murine bone marrow progenitors by retroviral infection, also induces acute myeloid leukemia (AML).

The transgenic mouse model also implicates E2A-PBX1 in the induction of apoptosis in lymphoid cells. The binding of both PBX1 and E2A-PBX1 to the consensus PBX1 DNA sequence is stimulated by direct interactions between PBX1 and other HOX proteins. Because HOX proteins appear to direct E2A-PBX1 to DNA sites recognized by HOX:PBX1 complexes, E2A-PBX1 likely interferes with hematopoietic differentiation by disrupting gene expression that is normally regulated by HOX proteins. In this regard, E2A-PBX1 can induce the aberrant expression of developmentally regulated genes when it is expressed in fibroblasts.

Surprisingly, B-cell precursors, the target of E2A-PBX1 in human leukemias, cannot be transformed in culture. Instead, the inducible expression of E2A-PBX1 in B-cell progenitors induces TP53- independent apoptosis, suggesting that the in vivo leukemogenic potential of E2A-PBX1 may depend on cell type-specific resistance to apoptosis.

Molecular characterization of the t(1;19) has led to the development of reverse transcriptase–polymerase chain reaction (RT-PCR) assays that detect the E2A-PBX1 chimeric transcript. These assays can detect E2A-PBX1 fusions in patients for whom the results of cytogenetic studies were normal or in whom studies were unsuccessful, and they can identify patients whose cells contain the t(1;19) but lack the fusion gene.

With intensified chemotherapy, event-free survival (EFS) estimates that were once about 50% are now closer to 80%; this increase suggests that the adverse prognostic impact of this fusion can be overcome with chemotherapy that is more effective.

MLL fusion genes

The MLL gene, located at band 11q23, is altered in approximately 80% of infant ALL cases, 3% of ALL cases involving older children, and 85% of secondary AML cases that arise in patients who have been treated with topoisomerase II inhibitors. MLL encodes a 431-kd protein that contains 3 AT hook domains at the N-terminus, 2 central zinc finger domains, a region with homology to DNA methyltransferases, and a C-terminal region that shows high homology to the Drosophila trithorax protein. In Drosophila, trithorax regulates various homeotic genes and is required for normal development. In human leukemias, 11q23 translocations cluster in an 8.5-kb region of MLL and fuse the N-terminal portion of MLL, containing the AT hook and methyltransferase domains, to over 25 different proteins.

Loss of MLL function has been studied using gene knockout techniques. MLL heterozygous mice are small at birth, demonstrate retarded growth, and display anemia and thrombocytopenia. MLL- deficient mice die in utero and fail to express specific HOX genes. Although gene knockout experiments suggest that the loss of one MLL allele may contribute to leukemogenesis, proof that MLL fusions directly contribute to leukemogenesis was derived from chimeric mice that express MLL-AF9 under the control of normal MLL transcriptional elements. After a latency period of 4-12 months, AML develops with great frequency in chimeric mice whose cells express MLL-AF9, and the leukemic phenotype is similar to that of patients carrying the t(9;11).

In contrast, leukemia does not develop in mice whose cells express a truncated MLL gene; this finding suggests that the fusion protein is essential for tumorigenesis. These experiments not only demonstrate that chromosomal translocations are directly involved in tumor development but also they provide a model system for studying other MLL fusion genes.

MLL rearrangements confer a dismal prognosis on infants with ALL; long-term EFS rates are approximately 20%. A subset of these patients, however, particularly those whose disease responds well to initial chemotherapy, have a relatively favorable outcome.

TEL-AML1 gene fusion

The t(12;21) is detected by karyotyping in fewer than 0.05% of ALL cases. However, molecular techniques have demonstrated that the TEL-AML1 fusion gene, created by the t(12;21), is present in approximately one fourth of childhood ALL cases. In the resulting chimeric protein, the helix-loop-helix (HLH) domain of TEL is fused to the DNA-binding and transactivation domains of AML1.

TEL and AML1 are involved in various other leukemia-associated translocations. TEL originally was cloned as a fusion of TEL with the gene encoding the platelet-derived growth factor receptor b (PDGFR b); this fusion was caused by the t(5;12) in chronic myelomonocytic leukemia. AML1 is the DNA-binding component of the AML1:CBF transcription factor complex, which is the most frequent target of myeloid-associated translocations, including t(8;21), t(3;21), and inv(16).

The TEL-AML1 has been proposed to transform cells by interfering with AML1- mediated expression of HOX genes involved in lymphopoiesis. In this regard, fusion of TEL to AML1 converts AML1 from an activator to a repressor of transcription; this repression depends on the HLH dimerization motif of TEL. The leukemogenic properties of TEL-AML1 (and the other TEL fusions) may also involve disruption of the normal TEL pathway as TEL-AML1 forms heterodimers with and inactivates TEL.

Although the targets of TEL are unknown, the role of TEL in normal development has been examined by the targeted disruption of TEL in mouse embryos. TEL- deficient mice die at approximately day 11 of embryogenesis because of defective yolk sac angiogenesis and apoptosis of neural and mesenchymal cells; this finding establishes TEL as an important regulator of embryologic development.

TEL-AML1 expression is associated with an excellent prognosis; EFS estimates approach 90%. Recent results indicate a 10-year cumulative incidence of relapse of only 9% ±5% for patients whose cells are positive for TEL-AML1. Thus, TEL-AML1 expression identifies a large subset of patients with B-precursor ALL who have a favorable outcome.

Activation of myc in B-cell ALL

Mature B-cell ALL is characterized by the presence of surface immunoglobulin (IG), morphology characteristic of the French-American-British (FAB) classification L3, and translocations involving the myc gene on chromosome 8, band q24.

Approximately 80% of B-cell cases contain the t(8;14)(q24;q32), in which myc is translocated to the IG heavy chain gene locus. Nearly all of the remaining cases contain the t(2;8)(p12;q24) or the t(8;22)(q24;q11), in which either the k (located at band 2p12) or l (located at band 22q11) light chain gene is translocated to a region that is adjacent to myc. All 3 translocations lead to increased myc expression.

In turn, altered interactions between the myc protein and several other transcription factors are thought to lead to lymphoid transformation. Normally, myc dimerizes with the MAX transcription factor, which also can form heterodimers with MAD and Mxi1. myc:MAX dimers activate gene expression, whereas MAD:MAX dimers interact with the Sin3A protein to repress transcription. Overexpression of myc as a result of the t(8;14) or related translocations leads to increased levels of myc/ MAX heterodimers relative to MAD:MAX, ultimately causing transformation by the activation of unknown target genes.

Although B-cell ALL does not respond well to the conventional chemotherapy used to treat childhood B-precursor ALL, outstanding results (EFS estimates of approximately 80%) have been obtained with treatments designed for Burkitt lymphoma, which emphasize cyclophosphamide and the rapid rotation of antimetabolites in high dosages.

Activation of transcription factor genes in T-cell ALL

Recurring translocations in T-cell ALL often involve the transcriptionally active sites of the TCR b locus (7q34) or the TCR a and TCR d locus (14q11); these translocations lead to dysregulated expression of transcription factor genes. Like the translocations identified in B-cell ALL, these rearrangements may result from mistakes in the normal recombination process involved in the generation of functional antigen receptors.

Transcription factor genes altered in T-cell ALL include members of the bHLH (myc, TAL1/SCL1, TAL2/SCL2, LYL1), LIM (LMO1/RBTN1/TTG1, LMO2/RBTN2/TTG2), and homeodomain (HOX11) families.

More than 50% of T-cell ALL cases have mutations that involve NOTCH1; this gene encodes a transmembrane receptor that regulates normal T-cell development. Aberrant NOTCH1 signaling may entail constitutive expression of myc and cooperation with RAS.


Genetics of Childhood Acute Myelogenous Leukemia

The development of acute myelogenous leukemia (AML) involves an arrest in the maturational process of granulocyte or monocyte precursors secondary to chromosomal translocations or other accumulation of other genetic abnormalities. Subtypes of AML, with corresponding identifiable genetic abnormalities and/or prognostic implications, have been classified according to 2 overlapping schema: the French-American-British (FAB) classification system, published 1976, [5] and the World Health Organization (WHO) classification system, published in 2002.

The FAB system is still in widespread use and focuses on cellular morphology and cytogenetic analysis. The relatively recent reclassification addressed the fact that the morphologic appearance, cytogenetic abnormalities, and clinical behavior of AML subtypes do not always correlate. The WHO criteria focus on the presence or absence of specific cytogenetic translocations and on whether the leukemia is associated with previous myelodysplasia. Some generalities can be made with regard to both genetic aberration and clinical prognosis across these classification schema. These mutations are summarized according to FAB subtype below.

  • FAB classification
    • M0 - Undifferentiated AML
    • M1 - AML with minimal maturation
    • M2 - AML with maturation
    • M3 - Acute promyelocytic leukemia
    • M4 - Acute myelomonocytic leukemia
    • M5 - Monocytic leukemia
    • M6 - Acute erythroid leukemia
    • M7 - Acute megakaryoblastic leukemia
  • WHO classification
    • AML with recurrent cytogenetic translocations
      • AML with t(8;21)(q22;q22) AML1/CBFalpha/ETO
      • Acute promyelocytic leukemia
      • AML with t(15;17)(q22;q12) and variants PML/RARalpha
      • AML with abnormal bone marrow eosinophils inv(16)(p13;q22) vagy t(16;16)(p13;q22) CBFbeta/MYH1
      • AML with 11q23 MLL abnormalities
    • AML with multilineage dysplasia
      • With prior MDS
      • Without prior MDS
    • AML with myelodysplastic syndrome
      • Alkylating agent–related
      • Epipodophyllotoxin-related
    • AML not otherwise categorized
      • AML minimally differentiated
      • AML without maturation
      • AML with maturation
      • Acute myelomonocytic leukemia
      • Acute monocytic leukemia
      • Acute erythroid leukemia
      • Acute megakaryocytic leukemia
      • Acute basophilic leukemia
      • Acute panmyelosis with myelofibrosis

Undifferentiated AML (M0)

The cytogenetics of this subtype of AML subtype are varied; 20% of cases harbor complex and unbalanced karyotypes, whereas 15-20% of cases demonstrate chromosomal rearrangements of chromosome 5, chromosome 7, or both. Another 15% of cases involve trisomy 8 or chromosome 11 rearrangements (11q23). Other known aberrations include trisomy 13, t(9;22)(q34;q11) or variable tetraploidy. Of note, approximately 25% display a normal karyotype.

AML with minimal maturation (M1)

Genetic abnormalities associated with AML-M1 are often random. Occasionally, the t(8;21)(q22;q22) translocation is present, although this translocation is more strongly associated with the M2 subtype. Translocations may be found at the 11q23 band, which contains the locus for the MLL gene, the human homologue of the drosophila trithorax gene. However, abnormalities at this locus are more commonly associated with AML subtypes M4 and M5, and the molecular consequences of MLL gene rearrangments are discussed above.

The t(16;21)(p11;q22) translocation occurs in AML-M1, as well as in AML-M2 and AML-M7, at low frequencies. Nonetheless, this translocation is of interest for 2 main reasons. First, the presence of this translocation confers a very poor prognosis; second, the gene product resulting from this translocation is structurally similar to the EWS-ERG fusion protein, which is found in 5-10% of Ewing sarcoma cases. The t(16;21)(p11;q22) translocation transpositions the ERG gene on chromosome 21 and the FUS gene on chromosome 16. FUS is a member of the TET family of transcription factors, which acts as adaptors between transcription and RNA processing. ERG polypeptide is one of the physiologic binding partner of FUS, and the t(16;21)(p11;q22) creates a chimeric fusion protein similar to the EWS-ERG protein. This chimeric protein has been shown to enhance the proliferative and self-renewal capacity of myeloid progenitor cells, possibly by upregulatingthe G-CSF gene.

AML with maturation (M2)

The AML1:CBFb transcription factor complex, also known as CBF, is the most common translocation target in human leukemia. It is disrupted in approximately 30% of AML cases and 40% of AML-M2 cases. AML1 is a member of the runt family of transcription factors and possesses DNA-binding, transactivation, and protein-protein interaction properties. Its DNA-binding affinity increases when it forms heterodimers with CBFb, which does not interact directly with DNA.

Knockout experiments have demonstrated that both AML1 and CBFb are essential for definitive hematopoiesis; these findings suggest that the AML1:CBFb complex regulates genes essential for normal blood cell development. The t(8;21)(q22;q22) translocation disrupts the AML1:CBFb complex. The t(8;21) creates an AML1-ETO fusion gene, the protein product of which includes the runt homology domain of AML1 fused to ETO.

Like AML1-CBFb, AML1-ETO binds DNA and interacts with CBFb; however, AML1-ETO dominantly represses normal AML1 -mediated transcriptional activation through interactions with the nuclear corepressor complex. ETO directly interacts with the nuclear corepressors N-CoR and Sin3A, forming a complex that recruits histone deacetylase (HDAC).

The AML1-ETO/N-CoR/Sin3A/HDAC complex leads to deacetylation of histones, alteration of chromatin structure, and active repression of AML1 target genes. Through these actions as a dominant negative protein complex, expression of AML1-ETO in the developing mouse produces a phenotype that is lethal to embryos and is similar to that caused by the loss of AML1. AML1-ETO also causes additional abnormalities in hematopoiesis that may represent preleukemic events.

This fusion protein binds to AML1 and transforms fibroblasts in vitro. Like AML1-ETO, CBFb-MYH11 also interferes with the normal transcriptional transactivation capacity of AML1-CBFb, in this case by binding and sequestering AML1 into inactive complexes.

Expression of this fusion protein in mice produces a phenotype similar to that of AML1-ETO mice, with abnormalities in early hematopoiesis. Recent data show that treatment of these mice with chemical mutagens produces a high frequency of AML; this finding suggests that cooperating genetic events are required for leukemic transformation by CBFb-MYH11.

The t(6;9)(p23;q34) may also be identified in AML-M2 and AML-M4; the CAN gene on chromosome 9 is translocated to the DEK gene on chromosome 6, producing the chimeric DEK/CAN protein. The function of this fusion is unclear because it appears to play a role in various signaling pathways, which may lead to apoptosis resistance and uncontrolled mitosis. This translocation is also associated with a poor prognosis.

Acute promyelocytic leukemia (M3)

The t(15;17)(q22;q21) is seen in 95% of AML-M3 cases and creates the well-characterized PML-RARa fusion gene. Most cases of acute promyelocytic leukemia (APL, AML-M3) are associated with a balanced translocation that involves the retinoic acid receptor-alpha (RARa) gene at band 17q21 and the PML gene at band 15q21. RARa is a ligand-dependent transcription factor that interacts directly with DNA to regulate many genes, whereas PML is a tumor suppressor that plays a role in apoptotic pathways. Normally, RARa binds DNA as a heterodimer with RXR and represses transcription by recruiting the N-CoR/Sin3A/HDAC corepressor complex, much like AML1-ETO.

Binding of ligand (retinoic acid) activates gene expression by causing disruption of this complex and the recruitment of coactivators. The PML-RARa fusion protein also inhibits transcription via the corepressor complex; however, unlike RARa, it is not activated by physiologic doses of retinoic acid; however, pharmacologic doses of all-trans-retinoic acid (ATRA) cause release of the corepressor complex and the recruitment of activators. Clinically, using ATRA to treat patients with APL causes terminal differentiation of leukemic promyelocytes and the induction of remission. The combination of ATRA and anthracycline-based chemotherapy has greatly improved the overall prognosis for these patients.

Of note, AML-M3 may be associated with numerous other translocations that also involve the RARa gene. These include t(11;17)(q23;q11), t(11;17)(q13;q11), t(5;17)(q31;q11), and t(17;17). Of these, only the t(11;17)(q23;q11) translocation creates a fusion protein (PLZF/RARa) that is not responsive to retinoic acid, an important prognostic factor.

Acute myelomonocytic leukemia (M4)

A strong association between AML-M4 and AML-M5 and deletion or translocations involving band 11q23 is observed. As stated above, 85% of secondary AML cases arising in patients who have been treated with topoisomerase II inhibitors are associated with alterations in this gene locus; the molecular pathogenesis is discussed above. In M4 eos, inv(16)(p13;q22) is frequently encountered and is virtually pathognomonic for this variant of AML-M4. Interestingly, the CBF gene is disrupted in this AML subtype by either inv(16) or t(16;16). These translocations result in the joining of the 5' portion of CBFb to the smooth muscle myosin heavy-chain gene (MYH11); this joining results in a chimeric CBFb-MYH11 protein. This genetic abnormality confers a favorable prognosis.

Acute monocytic leukemia (M5)

As stated above, AML-M5 is frequently associated with MLL gene arrangements. The most common translocations found in pediatric cases are the t(9;11)(p21;q23) and the t(11;19)(q23;p13.1) translocations, although more 50 other translocations involving this locus have been reported. The t(10:11) translocation confers a particularly poor prognosis.

Acute erythroid leukemia (M6)

AML-M6 is associated with no specific characteristic genetic abnormality; instead, heterogeneous karyotypes with numerous abnormalities are often found. Chromosomes 5 and 7 are frequently involved; therapy-related AML is modestly associated with deletion of chromosome 7q or 5q, whereas native AML-M6 is more frequently related to 5q del. The transforming factors associated with these genetic aberrations are unknown.

Acute megakaryoblastic leukemia (M7)

Similar to the AML–M6 subtypes, no specific genetic abnormality is strongly associated with AML-M7. Again, complex karyotypes with multiple abnormalities are the rule, and also involve deletions of 5q or 7q. Translocations involving the EVI1 gene, described above and associated with 3q21 or q26 aberrations are found in 25% of cases. In therapy-associated AML-M7, trisomy 19 and 21 may also be found.

Gene mutations in AML

FLT3 mutations, which involve internal tandem duplications, portend a poor prognosis in adults and children, but the incidence is appreciably lower in children. The prognosis of point mutations in FLT3 is less clear. They occur in 30-40% of acute promyelocytic leukemia. RAS and tyrosine kinase receptor mutations such as in ckit occur in 25% of AML. Prognostic significance is not clearly shown in children. GATA1 mutations are present in most if not all patients with Down syndrome patients with transient myeloproliferative disorder (TMD) or megakaryocytic AML. GATA1 is a transcription factor receptor for the development of erythroid, megakaryoctes, eosinophils and mast cells, and increase the sensitivity of cytosine arabinoside. NPM1 is linked to ribosomal protein assembly and transport, but the significance of mutation in this gene is not clear.


Genetics of Childhood Non-Hodgkin Lymphoma

Pediatric non-Hodgkin lymphoma (NHL) can be divided into 3 main categories, as follows: small noncleaved cell (SNCC) lymphoma, lymphoblastic lymphoma, and large cell lymphoma (LCL). SNCC lymphomas, including Burkitt lymphoma and Burkittlike lymphomas, have a mature B-cell phenotype and demonstrate the same translocations as those seen in B-cell acute lymphoblastic leukemia (ALL). The molecular genetics of B-cell ALL are described above. Most cases of lymphoblastic lymphoma are of T-cell origin and have the same type of genetic change seen in T-cell ALL, also described above. LCLs are subdivided into lymphomas of B-cell lineage, which include diffuse large cell lymphoma, follicular large cell lymphoma, and large cell immunoblastic lymphoma, which is mostly seen in adults and T-cell lineage and most notably includes anaplastic LCL (ALCL).

Burkitt lymphoma is a high-grade, monoclonal proliferation of B lymphocytes and has 2 major forms, the endemic (African) form and the nonendemic (sporadic) form. This neoplasm is typically extranodal; the endemic form most often involves the mandible or maxilla, and the nonendemic form typically involves the distal ileum, cecum, or mesentery. In approximately 85% of cases, Burkitt lymphoma involves reciprocal translocation of chromosomes 8 and 14 (t(8;14)(q24;q32)), which results in transposition of the c-MYC proto-oncogene on chromosome 8 with either the immunoglobulin heavy chain gene locus (IgH, IGH), the kappa light chain (IGK) gene locus, or the lambda light chain (IGL) locus.

In 80% of cases, the translocation occurs with the IGH locus. The c-MYC gene product is a helix-loop-helix transcription factor that functions as a critical regulator of mitosis, differentiation, and apoptosis by pleiotropically influencing DNA acetylation and gene expression. MYC is activated bymitogenic signals including Wnt, Shh, and EGF via the MAPK/ERK pathway, forms a heterodimer with the Max transcription factor, and is required for cellular entry into S-phase. Following transposition of the c-MYC gene, expressional dysregulation appears to exert a global effect of cell mitosis and apoptosis, leading to malignant transformation of the B-cells in which this translocation occurs.

Epstein-Barr Virus (EBV) is associated with approximately 95% of cases of the endemic forms of Burkitt lymphoma and 20-30% of the sporadic forms; recent evidence has begun to elucidate the mechanism by which latent infection of B lymphocytes promotes tumorigenesis. [6, 7] In memory B-cells with latent EBV infection, expression of Epstein-Barr nuclear antigen-1 (EBNA1) appears to exert a direct antiapoptotic effect within the cell. It also reduces expression of major histocompatibility complex (MHC) class I molecules and antigen-processing (TAPasin) proteins. Thus, latent EBV infection may promote B-cell tumorigenesis by altering the programmed cell death pathway, leading to cell survival even in the presence of chromosomal abnormalities (ie, translocations) and by facilitating tumor evasion of the immune system following transformation.

ALCL is a subtype of LCL commonly characterized by a T-cell phenotype, expression of the CD30 antigen, and an aggressive clinical profile with peripheral adenopathy and skin involvement. Because unambiguous morphologic or immunophenotypic criteria are lacking, diagnosing this entity can be difficult. Compounding the problem are similarities between ALCL and Hodgkin disease, which can lead to an erroneous diagnosis.

Approximately 90% of cases of ALCL harbor the t(2;5)(p23;q35), which fuses the region encoding the N-terminal portion of nucleophosmin (NPM) on chromosome 5 to the region encoding the tyrosine kinase domain of ALK on chromosome 2, creating an NPM-ALK chimera, a constitutively active tyrosine kinase. Overexpression of ALK in lymphoid cells appears to contributes to tumorigenesis by inappropriate phosphorylation of yet unidentified target proteins.

Reverse transcriptase–polymerase chain reaction (RT-PCR) assays have identified NPM-ALK fusion transcripts in many ALCL cases but not in cases of Burkitt lymphoma, lymphoblastic lymphoma, or Hodgkin disease. Thus, RT-PCR is a useful diagnostic tool in the diagnosis of LCL; however, not all cases of ALCL express NPM-ALK and not all tumors expressing this fusion transcript are classified as ALCL. The value of this assay increases considerably if NPM-ALK–positive cases are found to represent a clinically important subgroup of patients who require specific therapy.


Genetics of Childhood Solid Tumors

Effective clinical management of rhabdomyosarcomas, the family of tumors that includes Ewing sarcoma and primitive neuroectodermal tumor (PNET), and neuroblastomas depends on unequivocal diagnosis that can guide the selection of specific therapy. However, each of these tumors may first be seen as a soft-tissue mass with the appearance of undifferentiated small round cells. Although immunohistochemical analysis can help in the diagnostic workup for these tumors, this method has limitations. [8] Recently, molecular diagnostic techniques have had an important role in ensuring diagnostic accuracy. The identification of molecular alterations has important prognostic and therapeutic implications.


Most alveolar rhabdomyosarcomas contain 1 of 2 recurring translocations, namely, the common t(2;13)(q35;q14) or the rare t(1;13)(p36;q14). Both translocations disrupt the FKHR gene, which encodes a widely expressed transcription factor. The t(2;13) fuses part of the PAX3 transcription factor gene to FKHR, encoding a Pax3-Fkhr chimeric protein, whereas the t(1;13) creates a Pax7-Fkhr fusion. In vitro, these fusion proteins can function as transcriptional transactivators and can contribute to transformation. They enhance activation of target genes that include antiapoptotic Bcl-xl and suppress expression of TGFa2, FTI1, PDGF, and IGF1 receptors.

Reverse transcriptase–polymerase chain reaction (RT-PCR) assays have been developed to detect the chimeric transcripts resulting from these fusion events. Such tests are both specific and sensitive, enabling the detection of transcripts in as few as one tumor cell per 100,000 normal cells and identifying transcripts in cases that are not amenable to standard cytogenetic analysis.

Clinically, tumors expressing Pax7-Fkhr are associated with favorable features, and the prognosis for patients with these tumors is better than that of patients with Pax3-Fkhr –positive tumors. Embryonal sarcomas have allelic loss of 11p15.5 and overexpression of the IGF2 locus.

Ewing sarcoma and the primitive neuroectodermal tumors family of tumors

More than 90% of Ewing tumors are characterized by the EWS-FLI1 fusion gene formed by the t(11;22) or by variant EWS fusions caused by the t(21;22) or the t(7;22). The t(11;22) produces a chimeric transcription factor that includes the transcriptional transactivation domain of EWS fused to the DNA binding domain FLI1; this factor is presumed to function by the aberrant activation of target genes. RT-PCR and fluorescence in situ hybridization assays for this fusion have been useful in distinguishing Ewing sarcoma from other small round cell tumors.

The precise t(11;22) breakpoint location has recently been demonstrated to have possible prognostic significance. Two studies suggest that the more common type of breakpoint (designated type I) is associated with a favorable outcome. In vitro data reveals that the type I fusion produces a less effective transactivator than the type II fusion, which might explain a survival advantage in patients with the type I fusion.


Patients older than 1 year and those with tumor cell metastases have a poorer prognosis than other patients with neuroblastoma; these clinical features have been used to guide the selection of therapy. The identification of genetic alterations in this disease greatly improves risk assessment.

In contrast to sarcomas, which are characterized by genetic alterations that produce chimeric transcription factors, neuroblastoma is characterized by gene amplification, tumor suppressor inactivation, and alterations in gene expression.

Amplification of the MYCN oncogene, located on chromosome 2, band p24, occurs in about one fourth of tumors and is associated with advanced stage and rapid disease progression. In addition, MYCN amplification is a powerful predictor of outcome independent of stage and age and is therefore a factor used to assign patients to more intensive therapies. Loss of heterozygosity of the short arm of chromosome 1 is also associated with an unfavorable outcome, a finding suggesting that a tumor suppressor gene may be located in this region. Gain of all or part of chromosome 17 is the most common molecular finding although only unbalanced gains results in poor prognosis. In contrast, hyperdiploid tumors in infants with neuroblastoma respond favorably to standard therapy, whereas diploid tumors require more intensive treatment.

Finally, expression of neurotrophin receptors is highly correlated with both biologic and genetic features. For example, high TRKA expression is correlated with a lack of myc amplification and a favorable outcome. TRKB, however, is more commonly expressed in higher-stage tumors that also show myc amplification.

Current risk classification schemes rely on both clinical and biologic factors in an attempt to provide the appropriate intensity of therapy for each group of patients.


In contrast to Ewing sarcoma and rhabdomyosarcoma, recurring translocations and fusion oncogenes have not been identified in osteosarcoma. Instead, inactivation of tumor suppressor genes likely plays a role in the development of this tumor. Patients with germline mutations of either TP53 or RB1 are at increased risk of developing osteosarcoma, and loss of heterozygosity (LOH) at the sites of these genes (17p and 13q) is a frequent finding in tumors. In addition, 3q and 18q are common sites of LOH in osteosarcomas, suggesting that tumor suppressor genes located in these regions may be inactivated. Recently, increased expression of the growth factor HER2 has been associated with a poor response to chemotherapy and a worse outcome in osteosarcoma, providing both a prognostic marker and potential therapeutic target.

S ynovial sarcoma

Synovial sarcoma is the second most common soft tissue sarcoma. The main genetic change is t(X:18)(p11:q11), which fuses the SYT gene at 18q11 to either the SSX1 gene (xp11.23) or the SSX2 gene (Xp11.21); the type of fusion affects prognosis. Prognosis is poorer in the case of the former.

Brain tumors

Various tumor suppressor genes are implicated in the development of childhood brain tumors, including TP53 in brainstem gliomas and the PTEN gene in glioblastoma multiforme. However, the best-studied tumor is medulloblastoma, which is a PNET that arises in the cerebellum and is the most common brain tumor in children. Loss of chromosome 17p is the most common genetic abnormality in patients with medulloblastoma, occurring in as many as 50% of cases. Although most tumors arise sporadically, medulloblastoma also occurs in patients with Turcot syndrome (APC gene) and in those with Gorlin syndrome. The latter is characterized by developmental anomalies, radiation sensitivity, basal cell carcinoma, a propensity to develop medulloblastoma, and germline mutations in the PTC gene that produce a protein capable of binding the hedgehog family of signaling proteins. TRK-C is associated with a good prognosis andpromotesapoptosis. Epidermal growth factor receptor-2 (ERBB-2), PDGF receptor, and insulin-like growth factor receptor are all associated with a poor prognosis.

The following are suggested prognostic indicators of glioblastomas and other gliomatous subtypes: p53 mutation and expression, overexpression or amplification of EGFR, CDKN2A alterations and deletion, and MDM2 amplifications. MDM2 is the key to maintaining proliferation and apoptosis. LOH of 10q leads to a shorter survival in glioblastoma multiforme and LOH of 1p and 19q may afford more favorable prognosis.

Basal cell carcinomas from patients with Gorlin syndrome often demonstrate loss of the second PTC allele, suggesting that PTC functions as a tumor suppressor gene. In addition, one allele of PTC is occasionally mutated in sporadic medulloblastomas, implicating the PTC pathway in tumorigenesis. Interestingly, mice heterozygous for PTC also develop medulloblastoma, but the tumors retain one functional allele of PTC, indicating that haploinsufficiency of this gene is sufficient for oncogenesis.

Ependymomas are tumors composed of neoplastic ependymal cells that arise from the walls of the cerebral ventricles or the spinal canal. Cytogenetic studies suggest that ependymomas may represent a diverse group of tumors. Genetic abnormalities found in childhood ependymomas include loss of chromosome 22, alterations in chromosome 6, monosomy 13, and loss of heterozygosity of 17p.

Wilms tumor

Although more than 95% of Wilms tumor cases are sporadic, this disease can also occur in the context of congenital anomalies or as part of a familial predisposition syndrome. Patients with congenital anomalies or a family history often have bilateral tumors and are diagnosed at an earlier age, indicating the germline loss of a tumor suppressor gene in these children. Syndromes associated with Wilms tumor include Beckwith-Wiedemann syndrome (BWS), Denys-Drash syndrome of renal failure and genitourinary (GU) anomalies, and WAGR syndrome (Wilms tumor, aniridia, GU anomalies, and mental retardation). Cytogenetic studies of patients with WAGR syndrome and sporadic Wilms tumor demonstrated the importance of the 11p13 band in the development of Wilms tumor. This led to the cloning of the WT1 tumor suppressor gene. WT1 encodes a transcription factor that is important innormal kidney development and functions as a classic tumor suppressor.

However, mutations of WT1 are detected in a minority of sporadic Wilms tumor cases, suggesting that other genes are involved in the development of this disease. Aberrant expression of genes located at 11p15, such as H19, IGF2, and p57, as well as other loci, are also likely involved in tumorigenesis. In addition, a large number of anaplastic histology Wilms tumors contain p53 mutations. A significant correlation of WT1 mutations and B catenen mutations (a cellular adhesion protein) has been noted. Loss of heterozygosity for 16q, 1p, and 22q are associated with adverse outcomes.

Children with BWS are predisposed to Wilms tumor; these children are also at increased risk to develop hepatoblastoma, neuroblastoma, and rhabdomyosarcoma. In addition to predisposing persons to cancer, BWS is characterized by prenatal and postnatal gigantism, abdominal wall defects, macroglossia, and hemihypertrophy. BWS is usually sporadic, but autosomal dominant transmission has been reported as well. Both sporadic and hereditary forms have alterations of band 11p15. Uniparental disomy of 11p15 and H19 hypermethylation carries the highest tumor risk. This fact initially supported the hypothesis of a "BWS" gene in this region. However, BWS is likely caused by an imbalance in the expression of several genes in this region rather than by the disruption of a single gene.

Imprinting studies suggest that increased expression of paternally derived growth-promoting genes (potentially IGF2) or decreased expression of maternally derived suppressor genes (possibly H19 or p57) lead to the phenotypic variability in BWS. Further studies of these genes in BWS and in Wilms tumor should provide insights into the development processes involved in somatic overgrowth and tumorigenesis.

Hodgkin lymphoma

Classic Reed-Sternberg cells, the pathognomnemonic cells associated with Hodgkin lymphoma, typically display immunoglobulin (Ig) variable (VAR) region gene rearrangements. On the chromosomal level, various abnormalities may be found in virtually all cases, suggesting genomic instability, although no specific rearrangement has been recognized as prognostically important. A common phenotype of these cells, however, is that they are resistant to apoptosis, a key feature of many lymphomas.

On the molecular level, resistance to apoptosis may be related to elevated or constitutive activity of NF - κ B, a pro-survival transcription factor that renders cells resistant to CD95-induced apoptosis. Of note, somatic mutations of the NF-κ B inhibitor I κ B may be detected in as many as 30% of cases; I κ B normally sequesters dimers of NF-κ B in the cytoplasm, and loss of function permits NF-κ B to enter the nucleus. c-FLIP, the c-FLICE inhibitory protein, is upregulated by NF-



Characterization of the genes situated at translocation breakpoints in childhood tumors has provided new insights into the mechanisms of malignant transformation. These observations have also allowed the development of molecular diagnostic assays, which have had a tremendous impact on the treatment of childhood cancer.

Even these techniques have their limitations, and full characterization of the complex genetic changes in cancer cells requires novel methods, such as DNA microarray technology. Such technology is likely to lead to refinements of current classification schemes and to help to characterize the downstream targets of oncogenic transcription factors. In the future, these methods may also lead to the development of novel therapies, including drugs that block chimeric transcripts or that interfere with the modulation of gene expression by these proteins.