Chapter 443. Cancer Genetics and Biology

Cancer is the most common cause of disease-related death in children beyond the newborn period. The unique biological features, the cell of origin, and the response to therapy of childhood cancers make them intriguing models with which to study and understand the process of human carcinogenesis. Tumors of childhood commonly reflect the embryonic precursor of the cell types in which they arise. The majority occur sporadically, and their etiology remains unclear. Obvious environmental influences to cancer initiation are not generally apparent. Some cancers, such as neuroblastoma, appear to arise even before birth, whereas a significant number of others present with a striking family history of cancer or coincide with congenital abnormalities, suggesting an important role for inherited causal genetic factors. Disorders such as xeroderma pigmentosum,1 which places children at increased risk for developing skin cancers, or Beckwith-Wiedemann syndrome,2 which is commonly complicated by the development of embryonal tumors, including Wilms tumor of the kidney, rhabdomyosarcoma, or hepatoblastoma, are examples of inherited disorders that confer an increased risk developing cancer. Cancer predisposition syndromes in which nonmalignant phenotypic features are not observed include hereditary retinoblastoma,3 Li-Fraumeni syndrome,4 and familial polyposis,5 while others such as von Hippel Lindau disease6 are associated with the coincident presentation of both benign and malignant neoplasms.

With the advent of technologies that can provide highly sensitive genome-wide molecular analysis, as well as clinical oncologists and geneticists recognizing the value of carefully ascertaining family cancer histories, it is likely that both new cancer predisposition associations and novel cancer genes will continue to be discovered. Importantly, new genetic tests offer opportunities for presymptomatic diagnosis of syndromic associations and a greater role of clinical surveillance by pediatricians and family physicians for early cancer detection in high-risk individuals. Study of both sporadic childhood cancers and familial syndromes associated with childhood malignancies has led to the identification and functional characterization of numerous tumor suppressor genes (TSGs), oncogenes, and DNA repair genes. TSGs normally control cell proliferation by either inhibiting progression through the cell cycle or promoting apoptosis (programmed cell death). Inactivation of both alleles of a TSG by mutation or deletion causes uncontrolled proliferation, thereby contributing to tumorigenesis. In contrast, oncogenes normally potentiate cell proliferation, and mutation of one allele is sufficient to produce uncontrolled cell growth. Inactivation of DNA stability or repair genes permits cells that harbor damaged DNA to divide, potentially leading to malignant transformation of those cells.2 Nonrandom molecular and cytogenetic alterations are frequently observed in most childhood cancers. These markers are unique diagnostic identifiers and frequently provide prognostic value with respect to disease outcome and anticipated response to therapy. Importantly, many of these genetic markers also recapitulate normal developmental processes, and as such offer a window into the biological mechanisms of both carcinogenesis and normal embryologic growth and development (Table 443-1). This chapter addresses the diverse molecular mechanisms in several prototypical childhood cancers and cancer predisposition syndromes, highlighting the critical role of molecular and biological pathways in cancer formation.

Retinoblastoma (RB) is the prototype cancer caused by mutations of a tumor suppressor gene. RB is a rare childhood tumor thought to arise in the embryonic retinal epithelium.7 RB occurs in 1 in 15,000 to 20,000 births, with 90% arising in children under 3 years of age.3,8 This condition occurs in either a sporadic or a hereditary form. Patients with sporadic RB usually have unilateral tumors and are older (median age 22 months) at diagnosis, whereas patients with hereditary RB often develop bilateral or multifocal tumors and are younger (median age 11 months) at diagnosis.4 Based on this inheritance pattern, Knudson proposed the “two-hit hypothesis,” which established the basis for discovery of TSGs.5 According to this hypothesis, the inactivation of both alleles of the causally associated gene is necessary for tumor development. In hereditary tumors, one mutated allele is inherited through the germ line and the second one is inactivated by a somatic event, often a gene deletion. In sporadic tumors, both alleles are inactivated in the somatic cell, leading to their unifocal and later onset.

The RB1 gene was initially localized to chromosome 13 (13q14)6 and was the first TSG to be cloned.7 Loss of function of both alleles of RB1 in tumor specimens8 and the presence of germ-line mutations in hereditary retinoblastoma9 validated Knudson’s two-hit hypothesis. RB1 is mutated or deleted in all retinoblastomas and in a wide spectrum of other tumors.10 It is thought that several genetic mechanisms may be involved in eliminating the second wild-type RB1 allele in an evolving tumor. These include chromosomal duplication or nondisjunction, mitotic recombination, or gene conversion.11–16 The retinoblastoma protein, pRB, serves as the “molecular switch” that controls the passage of cells through division and proliferation.11,12 Loss of this regulatory function allows uncontrolled proliferation, thereby contributing to tumorigenesis.

In approximately 40% of retinoblastoma cases, the disease is inherited as an autosomal dominant trait, with a penetrance approaching 100%.9 The child inherits one mutant allele at the retinoblastoma susceptibility locus (RB1) through the germ line; an acquired somatic mutation in the remaining normal allele occurs in a single retinal cell, leading to tumor formation. The remaining 60% of retinoblastoma cases are sporadic (nonheritable),10 and both RB1 alleles in a single retinal cell are inactivated by somatic mutations. These patients usually present with one tumor later than do infants with the heritable form. Fifteen percent of unilateral retinoblastoma is heritable9 but by chance develops in only one eye. Survivors of heritable retinoblastoma have a hundredfold increased risk of developing mesenchymal tumors such as osteogenic sarcoma, fibrosarcomas, and melanomas later in life.10,11 While these tumors may occur within the radiation field used for treating the primary disease, they more frequently occur outside the radiation field in children who have received no chemotherapy, implicating the hereditary component in these nonocular tumors as well.12

RB1 encodes a 105-kD nuclear phosphoprotein.17,18 In addition to being altered in retinoblastoma, this gene and its protein product have been found to be mutated in a wide spectrum of cancers, including osteosarcoma; small cell lung carcinoma; and bladder, breast, and prostate carcinomas.19,20 The protein pRB plays a central role in the control of cell-cycle regulation, particularly in determining transition from G1 through the S (DNA synthesis) phase in virtually all cell types.21 This protein also suppresses growth by binding to the E2F family of transcription factors during the G1. These E2F transcription factors then contribute to the activation of several different genes encoding S-phase functions related to the synthesis of DNA, including dihydrofolate reductase (DHFR), the thymidine kinase gene, and DNA polymerase. When E2F is complexed to pRb, E2F-mediated transcriptional activation does not occur.21 E2F is bound to only the underphosphorylated (active) forms of pRb and becomes active only when Rb is inactivated by phosphorylation. As the cell progresses into the S phase, Rb becomes phosphorylated and is no longer able to inhibit E2F-mediated transcriptional activation.

In the developing retina, inactivation of the RB1 gene is necessary and sufficient for tumor formation.22 It is now clear, however, that these tumors develop as a result of a more complex interplay of aberrant expression of other cell-cycle-control genes. In particular, a tumor surveillance pathway mediated by Arf, MDM2, MDMX, and p53 (see below) is activated after loss of Rb1 during development of the retina.23 RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumor progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells.24

These observations provide a provocative biological mechanism for tumor formation in retinoblastoma, and they point to potential molecular targets for developing novel therapeutic approaches to this tumor.25 Although the RB1 gene is expressed in virtually all mammalian tissues, only in the retina is its inactivation sufficient for tumor initiation. On the other hand, some RB1 mutations appear to lead to an attenuated form of the disease, an observation that highlights the variable penetrance in families.26,27 Outside the retina, RB1 inactivation is often a rate-limiting step in tumorigenesis generated by multiple genetic events, and these defects—along with the observation that mice lacking the RB1 gene are viable only through day 16 of gestation,28 as well the occurrence of specific organ defects—suggest that the critical role of Rb may be in differentiation of these affected tissues rather than in the control of cell growth per se.29

The patterns of inheritance and presentation of retinoblastoma have been well described and the responsible gene identified. These provide a sound rationale on which genetic counseling is conducted for affected families.30,31 Both the transmissibility of the genetic trait and the likelihood of developing secondary tumors can be predicted with some confidence, offering opportunities for presymptomatic clinical surveillance, early detection, and intervention. Furthermore, evaluation of other family members is greatly facilitated once a particular mutation is identified in a proband. As the biological mechanisms by which RB1 gene inactivation are better understood, evidence indicates that intricate functional interactions of pRB with its binding partners and other cell-cycle targets will offer opportunities to explore development of novel small-molecule therapies to complement conventional approaches.

Wilms tumor (WT), or nephroblastoma, is an embryonal malignancy that arises from remnants of immature kidney.32 It affects approximately 1 in 7000 children, usually before the age of 6 years (median age at diagnosis is 3.5 years). Five to 10% of children present with either synchronous or metachronous bilateral tumors.33 The relationship between WT and aberrations of normal development is striking and suggests common genetic and developmental pathways of normal and tumorigenic development.

A peculiar feature of WT is its association with nephrogenic rests, the foci of primitive but nonmalignant cells whose persistence suggests a defect in kidney development. These precursor lesions are found within the normal kidney tissue of more than one third of children with WT. Nephrogenic rests may persist, regress spontaneously, or grow into a large mass that simulates a true WT, thus presenting a difficult diagnostic challenge.34–36 WT is often associated with specific congenital abnormalities, including genitourinary anomalies, aniridia, mental retardation, and hemihypertrophy. A genetic predisposition to WT is observed in two distinct syndromes with urogenital system malformations—the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, mental retardation) syndrome and the Denys-Drash syndrome (DDS)37—as well as in Beckwith-Wiedemann syndrome (BWS), a hereditary overgrowth syndrome characterized by visceromegaly, macroglossia, and hyperinsulinemic hypoglycemia (see below).38 These congenital disorders have now been linked to abnormalities at specific genetic loci implicated in Wilms tumorigenesis.

WAGR syndrome is associated with constitutional deletions of chromosome 11q13, which harbors both the aniridia gene Pax639,40 and the WT1 gene.39,41-43 WT1 spans approximately 50 kb of DNA and contains 10 coding exons. The identity of the gene(s) targeted by WT1 during normal kidney development is not known. The WT1 tumor suppressor gene is reduced to homozygosity,44,45 at least in part, in a small but highly informative set of sporadic WT. In addition, sporadic and hereditary WT have been described in which WT1 is specifically altered. While point mutations of WT1, usually nonsense or frameshift mutations, are not commonly detected, tumor-specific deletions at the WT1 locus have been identified and suggest that this allele is specifically inactivated during pathogenesis of some WTs.

The second syndrome closely associated with this locus is Denys-Drash syndrome (DDS),46,47 which presents as an association of Wilms tumor (WT), intersex disorders, and progressive renal failure.47 Virtually all patients with DDS carry germ-line WT1 point mutations.48 Germ-line WT1 mutations are also observed in children with hypospadias and other developmental anomalies of the male genitalia.

WT1 is altered in only 10% of sporadic Wilms tumors.45 This implies that other genetic loci are important in its etiology. One such locus also resides on the short arm of chromosome 11, telomeric of WT1, at 11p15. This gene, designated WT2, is associated with BWS. These patients are at increased risk of developing Wilms tumor and other embryonic malignancies, including rhabdomyosarcoma (RMS), neuroblastoma, and hepatoblastoma.2,49 The putative BWS gene maps to chromosome 11p1550 and is tightly linked to the Ha-ras oncogene homologue HRAS-I and the insulin-like growth factor II gene (IGF2). Other genes, including CDKN1C (p57KIP2), a maternally expressed gene that encodes a cyclin-dependent kinase inhibitor and negatively regulates cell proliferation,51 show aberrant methylation in tumors that are associated with cell-cycle deregulation. However, CDKN1C is rarely mutated in Wilms tumors.52 Using long-oligonucleotide array comparative genomic hybridization (array CGH), a novel gene termed WTX was identified on chromosome Xq11.1. WTX is inactivated in one third of WTs, and tumors with WTX mutations lack WT1 mutations. Whereas autosomal tumor suppressor genes undergo biallelic inactivation, WTX is inactivated by a monoallelic “single-hit” event that targets the single X chromosome in Wilms tumors in males and the active X chromosome in tumors in females.53 This observation suggests a more important role of the X chromosome in human cancers than had previously been appreciated.54 Bilateral WT or a family history of WT occurs in 1% to 5% of patients. Although linkage studies have indicated that the gene for familial WT must be distinct from WT1 and WT2 and from the gene that predisposes individuals to BWS, to date, this gene has been neither cytogenetically localized nor isolated. Whether the gene for familial Wilms tumor interacts with the gene product of either of the two Wilms tumor suppressor genes has yet to be determined.

Finally, loss of the long arm of chromosome 16 has been observed in approximately 20% of Wilms tumor samples.55 This observation implicates yet another genetic locus in Wilms tumor. Linkage studies have generally also excluded this locus as the “familial” Wilms tumor gene.56 However, it is plausible that alterations at 16q can initiate tumorigenesis or be implicated in subsequent steps in the progression of malignancy.

Although both tumors represent classic models of the Knudson two-hit hypothesis of tumor development, the spectrum of genetic alterations in Wilms tumor is quite different from that in retinoblastoma. In retinoblastoma, strong evidence exists that a single gene is involved whose function is mediated through related cell-cycle control pathway genes, whereas a series of genetic alterations, or at least distinct genetic events, is required for Wilms tumorigenesis. Additionally, unlike with retinoblastoma, which is not associated with other developmental anomalies or congenital abnormalities, patients with Wilms tumor commonly exhibit a spectrum of nonmalignant congenital abnormalities that suggest a dual role of the genes involved in both tumor formation and normal embryonic development.

Neuroblastoma (NB) is the most common tumor of the peripheral sympathetic nervous system in children. NB most commonly arises in cells of the adrenal medulla and at other abdominal retroperitoneal sites of the known peripheral nervous system. Nonrandom cytogenetic abnormalities are observed in more than 75% of neuroblastomas.57-59 The most common of these is deletion or rearrangement of the short arm of chromosome 1,59 although loss, gain, and rearrangements of chromosomes 10, 11, 14, 17, and 19 have also been reported. The high frequency of gains on 17q and losses on 1p36 and 11q23 has prompted considerable interest. Allelic losses indicate loss of function of as-yet-unknown tumor suppressor genes in these regions. It is believed that a TSG that lies on band p36 of chromosome 1 is critically important in the pathogenesis and aggressive nature of neuroblastoma. It has been shown that the loss of chromosome 1p is a strong prognostic factor in patients with neuroblastoma, independent of age and stage.60,61 Although it is as yet unclear what gene(s) in this region may be directly implicated in neuroblastoma development, aberrant expression of one candidate—p73, which is a member of the p53 tumor suppressor family—appears to play a role in neuroblastoma cell growth and chemoresistance.62 Alternative splicing of the p73mRNA gives rise to more than seven protein isoforms that differ in the coding sequences of the COOH terminus (TA-p73 α, β, γ, δ, ε, ζ, η.63,64 Three additional forms, ΔNp73α, ΔNp73β, and ΔNp73γ, are transcribed from an alternative promoter located in intron 3. Their protein products lack the NH2-terminal transactivation domain and are thus called ΔNp73. The full-length forms that contain the NH2-terminal transactivation domain are denoted TA. Higher expression levels of ΔNp73 are associated with an overall worse clinical prognosis, presumably due to the “antiapoptotic” properties of ΔNp73 and its ability to inactivate both TAp73 and p53.65,66

Two other unique cytogenetic rearrangements are highly characteristic of neuroblastoma—homogeneous staining regions and double-minute chromosomes.67,68 These contain regions of amplification of the N-myc gene, an oncogene with considerable homology to the cellular proto-oncogene c-myc. N-myc amplification is associated with rapid tumor progression, and virtually all neuroblastoma tumor cell lines demonstrate amplified and highly expressed N-myc.69 Expression of N-myc is increased in undifferentiated tumor cells compared with much lower (or single-copy) levels in more differentiated cells (ganglioneuroblastoma and ganglioneuroma). Furthermore, a close correlation exists between N-myc amplification and advanced clinical stage.70On the other hand, decreased N-myc expression is observed in association with the in vitro differentiation of neuroblastoma cell lines.71 This observation formed the basis for current therapeutic trials demonstrating a survival advantage for patients treated with cis-retinoic acid.72

Neuroblastoma cells that express the high-affinity nerve growth factor receptor trkA73 can be terminally differentiated by nerve growth factor and can demonstrate morphological changes typical of ganglionic differentiation. Tumors showing ganglionic differentiation and trkA gene activation have a favorable prognosis.73 Resistance to multidrug chemotherapeutic regimens (multidrug resistance) is characteristic of aggressive, poorly responsive N-myc–amplified neuroblastomas. Expression of trkB receptor is associated with poor-prognosis tumors and appears to mediate resistance to chemotherapy.74,75 It is interesting to note that expression of the multidrug-resistance-associated protein (MRP), found to confer multidrug resistance in vitro, is increased in neuroblastomas with N-myc amplification and decreases after differentiation of tumor cells in vitro.76 High levels of MRP expression are significantly associated with poor outcome, independent of N-myc amplification.76 While gain of chromosome segment 17q21-qter has been shown to be the most powerful prognostic factor yet,77 no gene has currently been implicated at this site.

In addition to chromosomal loss on chromosome 1p36, unbalanced loss of heterozygosity at 11q23 is independently associated with decreased event-free survival. Alterations at 11q23 occur in almost one third of neuroblastomas, being most commonly associated with stage IV disease and age at diagnosis greater than 2.5 years. Both 1p36 LOH and 11q23 LOH were independently associated with decreased, progression-free survival in patients with low- and intermediate-risk disease.78,79 Telomerase expression and telomere length are other valuable markers of clinical significance. In particular, short telomere length is predictive of favorable prognosis, irrespective of disease stage, while long or unchanged telomeres are predictive of poor outcome.80,81 Telomerase expression, as measured by telomerase reverse transcriptase (hTERT), has been shown to be negative in “good risk” neuroblastoma while it is high in tumors with unfavorable histology.81 The combined use of these markers—LOH at chromosomes 1p36 and 11q23, N-Myc amplification, trkA, and telomerase expression—as prognostic indicators provides a powerful set of tools with which to develop rational stratified treatment programs for neuroblastoma.

While most neuroblastomas are sporadic in nature, study of rare cases of familial neuroblastoma led to discovery of a familial neuroblastoma gene (REF). Remarkably, this gene (identified using high-throughput genome-wide microarray analysis) is ALK, the tyrosine kinase associated with the t(2;5) translocation characteristic of anaplastic large-cell lymphoma. Activating mutations of the ALK gene induce proliferation of malignant neuroblasts. Furthermore, somatic mutations of this gene are observed in sporadic neuroblastomas as well, suggesting its importance in the genesis of this tumor.

The advent of molecular markers as indicators of tumor biology and outcome in NB may ultimately explain its varied clinical presentations, outcomes, and biological behavior. For example, a unique presentation of NB, stage IV-S (IV-special), is frequently associated with spontaneous remission.82,83 This form of the disease typically presents in infants with evidence of remote disease in the liver and bone marrow, though sparing bone. It is not associated with NMyc amplification or loss of chromosome 1p or other poor molecular markers. Whether IV-S NB represents metastatic disease or is a multifocal nonclonal disorder of neuroblast development is not known. The biological distinction is important, as patients with similarly presenting disease can also go on to develop more aggressive NB, suggesting that while the IV-S NB cells may have some limited metastatic potential, it is not entirely absent and may reflect subtle distinctions in genetic profile of the different cell types.

While NB is not a common human malignancy, its devastating prognosis and its wealth of biological and molecular features make it an ideal candidate for extensive and intensive study. The use of tyrosine kinase inhibitors to block the function of aberrant ALK expression offers hopeful opportunities for novel effective therapies for this disease.

The Ewing sarcoma family of tumors (ESFT) is the second most common bone malignancy after osteosarcoma in children and young adults with a peak incidence at age 15. In rare cases, these tumors can arise in the soft tissues. ESFT includes Ewing sarcoma, peripheral primitive neuroectodermal (PPNET), and Askin tumors. It is thought that these are most certainly identical tumors that are distinguished from each other only in the degrees of morphological “neuroectodermal” differentiation they exhibit when examined under light microscopy. They share indistinguishable genetic alterations, immunohistochemical profiles, and lineage-specific marker expression patterns. A striking distinguishing chromosomal translocation, t(11;22)(q24;q12),84-88 is frequently observed in peripheral primitive neuroectodermal tumor/Ewing sarcoma (PPNET/ES), though translocation-negative tumors are described.

Both PPNET- and ES-derived cell lines and tumors typically carry a t(11;22)(q24;q12) chromosomal rearrangement, although variant translocations have been observed. The translocation breakpoint is an in-frame fusion between the 5' half of the ES gene, EWS, on chromosome 22 and the 3' half of the human homologue of an ETS transcription family member, Fli-1, on chromosome 11.89-91While the gene that EWS encodes is ubiquitously expressed, FLI1 exhibits a much more restricted pattern of expression. The resultant chimeric protein replaces the DNA-binding domain of EWS with the ETS-like binding domain of Fli-1, retaining the DNA-binding activity of Fli-1.91 Targets of the EWS-Fli-1 transcription factor may include insulin-like growth factor I receptor (IGF-IR),92,93 which is thought to play a role in the pathogenesis of ES. Autocrine stimulation of the insulin-like growth factor I receptor (IGF-IR) is important for cell transformation and proliferation induced by EWS-Fli-1. Expression profiling analysis has also revealed that the p53 tumor suppressor is transcriptionally upregulated by the EWS-ETS fusion gene.94 This observation is intriguing, because expression of EWS-ETS can lead to apoptosis, and additional alterations, such as loss of p53 or p16 signaling, or both, appear to be necessary components of EWS-ETS-induced transformation.95 Using RNA interference technology to inhibit EWS-Fli-1 in ES cells to identify genes regulated by the fusion, another gene, NKX2.2, has been found to be an additional target gene of EWS-Fli1 that is required for malignant transformation.96

Several variant translocations have also been identified, invariably fusing the EWS gene to an ETS family member.97,98 Greater than 90% of the ES family of tumors (ESFTs) carry the EWS-ETS fusion gene, and a search for EWS-ETS by either reverse transcriptase-polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH) is considered standard practice in the diagnostic evaluation of suspected ESFTs. The specific fusion protein expressed in ESFT has prognostic significance99,100; specifically, a rearrangement that joins exon 7 of EWS to exon 6 of FLI1 may confer a more favorable outcome.101

Biochemical and molecular features of peripheral primitive neuroectodermal (PPNET) and Ewing sarcoma (ES) tumors may distinguish these from NB cells that otherwise have similar histological features. For example, MIC2 expression is easily detected in PPNET and ES cells, whereas it is not expressed in NB cells.102 The neuronal derivation of these tumors is substantiated by the fact that treatment with cyclic AMP or TPA (12 tetradecanoylphorbol-13-acetate) results in ES cells developing elongated processes that sometimes include neurofilaments, microtubules, and dense core neurosecretory granules.103 While nerve growth factor does not induce morphological differentiation of ES cell lines, nerve growth factor receptor and other members of the TRK gene family are frequently expressed in ES.104 IGF-I may be important in regulating proliferation of PPNET tumor cells.105 Most PPNET lines express IGF-I, and secretion of biologically active IGF-I and IGF-binding protein (IGFBP-2) has been demonstrated. Blockade of the IGF-IR by a monoclonal antibody known to interrupt ligand binding inhibits serum-free growth, indicating that IGF-I can be an autocrine growth factor for PPNET.105 While the EWS-Fli-1 fusion protein presents a tantalizing opportunity for development of novel molecular therapies, its sequestration in the nucleus makes it challenging to create effective agents (such as antibodies) that will target the nuclear fraction. Thus, inhibitors of IGF-I signaling appear to hold more promise.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, accounting for nearly 10% of all childhood solid tumors.106-108 Rhabdomyosarcoma is believed to arise from primitive embryonic mesenchymal cells committed to the skeletal muscle lineage; however, RMS tumors have been found in tissues not usually containing striated muscle, such as the urinary bladder.108 RMS cells express muscle-specific proteins such as myoglobin and creatine kinase, as well as tissue-specific basic helix-loop-helix transcription factors, including MyoD and Myf-3, which mediate the expression of a complex of genes responsible for muscle differentiation. RMS is a morphologically and biologically heterogeneous group of tumors. Multiple histological subtypes exist, including embryonal (ERMS; 63%) and alveolar (ARMS; 19%) morphologies. The alveolar subtypes tend to occur in the extremities and exhibit a more aggressive clinical behavior than their ERMS counterparts, which tend to present in an axial distribution and with a somewhat more favorable prognosis. The axial tumors (head and neck, bladder, prostate, paratesticular) are less frequently metastatic at the time of presentation, which is likely a significant factor in their more favorable outcome.

Characteristic genetic lesions have been found in both major RMS subtypes. More than 75% of ARMS demonstrates one of two chromosomal translocations, t(2;13)(q35;q14) or t(1;13)(p36;q14)109-111; these yield fusion between the 5' DNA-binding region of PAX-3 on chromosome 2 or PAX-7 on chromosome 1, respectively (implicated in neuromuscular development), and the 3' transactivation domain region of FKHR (FOXO1A) gene (a member of the forkhead family of transcription factors commonly associated with regulation of apoptosis).112 Fusion-negative ARMS are also described, and these appear to have an intermediate prognostic pattern between translocation-positive ARMS and ERMS. Tumors with the t(2;13) translocation have a much poorer prognosis than those with the less frequent t(1;13) rearrangement. To date, attempts to generate mouse models of alveolar RMS using knock-in of the PAX-3-FKHR fusion have led to developmental abnormalities but not to tumor formation.113 This implies other epigenetic or genetic events are required for malignant transformation, suggesting a multihit model for tumorigenesis in RMS. The biochemical interactions and associations of the PAX-FKHR fusions support this hypothesis. One such association is that of PAX-3-FKHR fusion with increased expression of c-Met.114,115 Met is the receptor tyrosine kinase for hepatocyte growth factor/scatter factor and is overexpressed in embryonal and alveolar RMS.116 A mouse model of embryonal RMS has been generated by expressing a hepatocyte growth factor transgene in Ink4a/Arf–/– mice. The tumors appear to arise from hyperplastic satellite cells (myoblastic precursor cells).117 The putative role of satellite cells in the pathogenesis of embryonal RMS is supported by a report demonstrating high PAX-7 expression in embryonal RMS compared to alveolar RMS and the association of PAX-7 expression with satellite cells.118 Other xenograft models of RMS have been generated,119-123 and while they are of particular value in exploring cell-signaling pathways in these tumors or in the use of novel therapeutic agents, they do not resemble primary genetic models of the disease.

Other genetic models of rhabdomyosarcoma (RMS) have been generated, including those expressing constitutively active Ras (V12G), SV40 T/t antigen, and hTERT, which develops characteristic embryonal RMS, and the p53-her2 mouse,124 which develops embryonal RMS. Other frequently reported genetic alterations that may be common to embryonal and alveolar RMS include activated forms of N- and K-RAS,125,126 inactivating TP53 mutations,127 and amplification and overexpression of MDM2, CDK-4, and N-MYC.127

The genetics of ERMS tumors are distinct from ARMS primarily in that they display a specific loss of heterozygosity (LOH) at chromosome 11p15, involving the loss of maternal and duplication of paternal genetic material. Classically associated with this LOH is the overexpression (due to a failure of imprinting) of the insulin-like growth factor II (IGF-II), which may be involved in the tumorigenic cascade that leads to ERMS. Other molecular alterations include dysregulation of the TP53 and retinoblastoma tumor suppressors, as well as the potent oncogene RAS.128-130

The LOH at 11p15 occurs by loss of maternal and duplication of paternal chromosomal material.131 Although LOH is normally associated with loss of TSG activity, in this instance, LOH with paternal duplication may result in activation of IGF-2. This occurs because IGF-2 is normally known to be imprinted. The gene is normally transcriptionally silent at the maternal allele, with only the paternal allele being transcriptionally active.132,133 Thus, LOH with paternal duplication potentially leads to a twofold gene dosage effect at the IGF-2 locus. Furthermore, in alveolar tumors in which LOH does not occur, the normally imprinted maternal allele is reexpressed.134,135 Thus, LOH and loss of imprinting may lead to the same functional result, namely, biallelic expression of the normally monoallelically expressed IGF-2. However, loss of an as-yet-unidentified tumor suppressor activity due to LOH also remains a possibility.

In addition to the somatic molecular changes associated with RMS, the tumor is also frequently observed in Li-Fraumeni syndrome (see below), in which carriers harbor constitutional mutations of the TP53 tumor suppressor gene. The possible importance of the patched gene, PTCH, in the development of RMS is suggested by the finding that mice lacking this gene develop RMS.136 PTCH is mutated in the germ line of patients with Gorlin syndrome, a disorder that includes predisposition to tumor (medulloblastoma) development. Strikingly, PTCH is shown to regulate another gene, GLI, which is found to be amplified in RMS and Gorlin syndrome–associated tumors.137

Rhabdomyosarcomas are generally exquisitely sensitive to radiation and chemotherapy. Managing these tumors therefore typically includes local control with both surgery and radiation treatment, with neoadjuvant chemotherapy used to control known and micrometastatic disease. It is notoriously difficult to achieve a sustained remission or cure for children with metastatic RMS, particularly those with the alveolar variant. Therefore, it is of great importance to identify tumor-specific molecular alterations and growth regulatory pathways that may provide rational targets for novel therapies. Whether these approaches will be most effective in blocking IGF-II-mediated signaling or in developing kinase inhibitors or agents that block proangiogenic pathways remains to be determined.

Osteosarcoma (OS) occurs most frequently in adolescence during a period of rapid bone growth.138 It is the most common solid tumor in this age group. OS usually appears at metaphyseal growth plates of long bones, develops earlier in girls than in boys, and is more frequent in taller children.139,140 These observations suggest an important role for cellular proliferation in the oncogenic conversion of immature bone precursors (osteoblasts) from which these tumors are thought to arise. One classification scheme for bone tumors is based on the tissue-specific differentiation of tumor cells.141 Tumors are classified as osteoblastic, chondroblastic, or fibroblastic, depending on whether the predominant differentiation is a long bone, cartilage, or stromal tissue pathway, respectively. These distinctions provide some insight into the molecular and biological behavior of individual tumors. The distinction between metastatic OS and multifocal bone OS is often difficult, reflecting different biological bases for their etiology.

The molecular basis of osteosarcoma (OS) is strikingly more complex than that of other bone tumors, such as ES/PPNET. In particular, OS is typically characterized by the presence of complex unbalanced karyotypes in most cases.142 Combined inactivation of the RB1 and p53 tumor suppressor pathways are observed in most OS, indicating important roles for both these genes in OS pathogenesis.143 Further evidence for the role of p53 in OS pathogenesis includes the predisposition of patients with germ-line TP53 mutations (Li-Fraumeni syndrome) to develop OS.144,145 In addition, transgenic mice expressing high levels of mutant TP53 also develop osteosarcomas at a high frequency, providing evidence of a key role for the TP53 gene product in regulation of bone growth. Modifying effects of other expressed genes are being explored in OS. In particular, amplification of chromosome 12q13 region (containing MDM2 and CDK4) or deletion of INK4A can detrimentally affect both the p53 and Rb pathways.146 There is low prognostic significance of TP53 mutations in sporadic OS, with no impact on distant recurrence. Furthermore, p53 status is concordant in paired samples of primary and distant metastases, suggesting p53 pathway alterations may occur early in OS pathogenesis.

Aside from p53 and Rb, few other TSGs or oncogenes have been definitively linked to the genesis of osteosarcoma. However, further study into the complex karyotype has yielded insight into other regions of the genome that may be of significance. Most osteosarcoma (OS) demonstrate marker chromosomes, structurally abnormal chromosomes in which no part can be identified, ring chromosomes accompanied by multiple numerical and structural abnormalities, and genomic amplifications. Common numerical abnormalities in OS include gain of chromosome 1; loss of chromosomes 9, 10, 13 (at the RB1 locus), or 17 (at the p53 locus); and partial or complete loss of the long arm of chromosome 6.147-150 Frequent structural abnormalities include rearrangements of chromosomes 11, 19, and 20.147,148,150-152 Genome-wide efforts to identify potential tumor suppressor genes associated with the loss of heterozygosity (LOH) in OS are under way.153,154 Examination of 38 chromosomal arms from OS tumor samples for LOH has found that the mean frequency of LOH is 30.79% for any chromosome arm, an unusually high mean frequency for a childhood tumor. Moreover, several chromosome arms (3q, 13q, 17p, and 18q) underwent LOH, with a frequency more than two standard deviations higher than the average (p < 0.002).153 Further mitotic mapping has identified minimal regions thought to contain candidate tumor suppressor loci on chromosomal arms 3q26.2 and 18q21.33.155,156 Further analysis has suggested that other chromosomal regions may also harbor tumor suppressor loci important in osteosarcoma tumorigenesis, including chromosome arms 5q, 6q, 10q, 11p15q, 16p, and 22q.157

Karyotypic complexity may reflect chromosomal fusion-bridge-breakage cycles that occur due to advanced telomere erosion. A potential etiology of this chromosomal instability is telomere dysfunction, as has been implicated in epithelial cancers.158 Telomeres are nucleoprotein structures that cap chromosome ends and serve at least three protective functions: preventing chromosomes from being recognized as damaged DNA, preventing chromosomal end-to-end fusions and recombinations, and accommodating the loss of DNA that occurs with each round of replication. Normal human somatic cells have finite proliferative capacity, and it has been demonstrated that telomere length is one of the checkpoints that determines when a cell stops dividing.159,160 As cells divide, the telomere length gradually decreases to a critical size, at which point senescence is triggered by a p53-dependent process.

Human cells may bypass this checkpoint by inactivating the p53 pathways, in which case they continue to divide until telomeres become very short, chromosomal instability ensues, and apoptosis is triggered. Rare cells bypass this second checkpoint by activating mechanisms that lengthen telomeres, a central feature of cancer cells. About 85% of cancers activate an enzyme called telomerase, which lengthens telomeres, and the other 15% of cancers use a recombination-based method called alternative telomere lengthening (ALT).161,162 Unlike most cancers, at least 50% of OS samples are dependent upon the ALT mechanism to maintain telomeres.163-165 The ALT and telomerase mechanisms are different means to the same end, but they are not equivalent. This is evident in the pattern of telomere size resulting from each mechanism. Telomerase-dependent OS cell lines have short telomeres with a minor range of length, whereas ALT-dependent OS cell lines have long telomeres with great heterogeneity in length. The ALT cell lines also have greater genetic instability and more translocations than the telomerase-positive cell lines.163 A mouse model of ALT indicates that ALT-dependent tumors had decreased metastatic potential compared to telomerase-dependent tumors.166 It has been hypothesized that ALT-dependent human OS has different clinical behavior than telomerase-dependent OS, but this remains to be studied.

Further insight into the molecular mechanisms leading to osteosarcoma (OS) is derived from studies of Rothmund-Thomson syndrome (RTS), a very rare disorder characterized by the presence of a very distinct rash termed poikiloderma, which begins in infancy, and skeletal dysplasias, including radial ray abnormalities and cataracts. Children with RTS have a distinct predisposition to the development of osteosarcoma and, less frequently, skin cancers. RTS is associated with germ-line mutations in the RECQL4,167 a member of the helicase family of genes that are involved in the process of chromatin remodeling. RECQL4 mutations appear to be rare in sporadic OS.

Abnormalities of bone growth and remodeling are thought to play an important role in the pathogenesis of OS. Normal bone repair involves proliferation of mesenchymally derived precursor cells mediated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), IGFs, interferon-γ, and other mitogens. The PDGF receptor encodes a tyrosine kinase, and when PDGF binds to its receptor, it induces expression of fos, myc, and a cascade of cellular genes important for initiating proliferation. EGF may also play a role in OS pathogenesis. Some OS cell lines express EGF receptor and proliferate in response to exposure to EGF. The mitogenic response to both EGF and PDGF may be blocked by TGF-beta, which at low doses may, in fact, stimulate cell proliferation.

Recent interest in other signaling pathways has implicated the Fas cell death pathway in determining chemosensitivity and metastatic behavior in OS.168,169 Fas is a type I transmembrane protein that regulates immune cell apoptosis, but Fas is expressed in many different cells and tissues and is functional in cells outside of the immune system.170 Tumor cells expressing surface Fas will apoptose when Fas ligand (FasL) is present unless a mechanism of resistance is present. Fas signaling may be involved in OS metastases.171 This has been suggested by studies in which metastasis-prone OS cell lines, when transfected with Fas, demonstrate reduced metastatic potential.172 How this pathway may be harnessed for therapeutic benefit is still to be determined. To date, biological response modifiers, monoclonal antibodies, and targeted small molecule kinase inhibitors have not impacted treatment of OS—a disease in which long-term outcome has not changed significantly in the last two decades.

Several hereditary cancer syndromes are associated with the occurrence of childhood and adult-onset neoplasms. This chapter discusses a few to highlight the important molecular basis on which these disorders form (Table 443-2).

Li-Fraumeni Syndrome

Li-Fraumeni syndrome (LFS) is the prototypical familial cancer predisposition syndrome. It was first described in 1969 from an epidemiological evaluation of more than 600 medical and family history records of children diagnosed with sarcoma.173,174 The definition of classical LFS requires a proband with a sarcoma diagnosed before 45 years of age and a first-degree relative with any cancer under 45 years of age and a first- or second-degree relative with any cancer under 45 years of age or a sarcoma at any age.4 The classical spectrum of tumors that includes soft-tissue sarcomas, osteosarcomas, breast cancer, brain tumors, leukemia, and adrenocortical carcinoma (ACC) has been overwhelmingly substantiated by numerous subsequent studies175; however, other cancers, usually of particularly early age of onset, are also observed.176 Families with similar cancer patterns that do not meet the classical definition have been coined Li-Fraumeni-like syndrome (LFS-L).177 While several “definitions” have been proposed for LFS-L, the description by Chompret and colleagues appears to most effectively resolve the molecular correlations: (1) a proband with a characteristic LFS tumor (sarcoma, brain tumor, breast cancer, adrenocortical carcinoma) before 36 yearsand at least one first- or second-degree relative with a characteristic LFS tumor (other than breast cancer if the probandis affected by breast cancer) before 46 years; (2) a proband with multiple primary tumors, two of whichare characteristic LFS tumors and the first of which occurredbefore 36 years, whatever the family history; or (3) a proband withadrenocortical carcinoma regardless of age of onset and familyhistory.178 Patients with LFS do not manifest any apparent nonmalignant stigmata.

Germ-line alterations of the TP53 tumor suppressor gene are associated with LFS.179-181 These are primarily missense mutations that yield a stabilized mutant protein. The spectrum of germ-line TP53 mutations is similar to that of somatic mutations found in a wide variety of tumors. Carriers are heterozygous for the mutation, and in tumors derived from these individuals, following Knudson’s two-hit model, the second (wild-type) allele is frequently deleted or mutated, leading to functional inactivation.182-184 Several comprehensive databases document all reported germ-line (and somatic) TP53 mutations and are of particular value in evaluating novel mutations and phenotype-genotype correlations.185 Approximately 75% of “classic” LFS families have detectable TP53 alterations. It is not clear whether the remainder are associated with the presence of modifier genes, promoter defects yielding abnormalities of p53 expression, or simply the result of weak genotype-phenotype correlations (ie, the broad clinical definition encompasses families that are not actual members of LFS). Other cancer predisposition genes, such as p16, p15, p21, BRCA1, BRCA2, and PTEN, associated with multisite cancer associations have generally been ruled out as potential candidates.

Germ-line TP53 alterations have been reported in patients and families with an incomplete LFS phenotype. Between 3% and 10% of children with apparently sporadic rhabdomyosarcoma or osteosarcoma carry germ-line TP53 mutations.186-189 These patients tend to be younger than those who harbor wild-type TP53. More than 75% of children with apparently sporadic adrenocortical carcinoma harbor germ-line TP53 mutations, although in some of these cases, a family cancer history frequently evolves with time.190,191 A unique genotype-phenotype correlation has been observed in a subgroup of ACC patients in Brazil in whom the same germ-line TP53 mutation at codon 337 has been observed in apparently unrelated kindreds.192 Other cancers typical of LFS are not observed in these families, and the functional integrity of the mutant protein appears to be regulated by alterations in cellular pH,193 which suggests potential biological mechanisms in ACC cells by which the p53 mutation leads to malignant transformation. These observations suggest that germ-line TP53 alterations may be associated with early onset development of the childhood component tumors of LFS.194 In light of the critical role played by p53 in the initiation and potentiation of irradiation or chemotherapy-induced DNA damage repair, studies into the effect of such germ-line mutations on the potentiation of tumor development related to therapeutic interventions would be important.

The variability in type of cancer and age of onset within and between LFS families suggests that expression of modifier genes might influence the underlying mutant TP53 genotype. Nonsense, frameshift, and splice mutations are commonly associated with particularly brain tumor–predominant LFS kindred.177 Missense mutations in the p53 DNA binding domain are frequently observed in the setting of breast and brain tumors, while adrenocortical cancers are associated with mutations in the non-DNA binding loops. Age of onset modifiers have also now been established. The protein murine double minute-2 (MDM2) is a key negative regulator of p53 and targets p53 toward proteasomal degradation. The MDM2 single nucleotide polymorphism (SNP) 309 increases Sp1 transcription factor binding, leading to increased MDM2 expression levels. Coinheritance of the MDM2 SNP309 T>G isoform is associated with earlier-onset cancer.195,196 The earlier age of onset of cancers with subsequent generations in mutant TP53 LFS families suggests genetic anticipation. This observation may be explained by accelerated telomere attrition from generation to generation, a molecular marker that may be of value in predicting age of tumor onset.196 Thus, while germ-line TP53 mutations establish the baseline risk of tumor development in LFS, a complex interplay of modifying genetic cofactors likely define the specific phenotypes of individual patients.

The primary treatment of patients with LFS is directed to the specific tumors. Discussions regarding the effect of chemotherapy or radiation therapy on the transforming effects of underlying TP53 mutants and the potential to accelerate the development of secondary malignancies are theoretical at best. Most conventional chemotherapeutic agents—such as the anthracyclines, alkylators, and platinum-based agents—and therapeutic doses of radiation induce cell growth arrest or apoptosis through a p53-mediated signaling pathway. Thus, to modify therapy in the face of mutant p53 is challenging and would severely limit the selection of effective anticancer therapy. For this reason, several approaches to developing wild-type gene replacement therapy or small molecules that enhance expression of wild-type p53 are under way. How these agents find utility in clinical practice remains to be determined. With the identification of germ-line TP53 mutations in asymptomatic children or young adults, it becomes possible to consider introducing aggressive clinical surveillance protocols to enhance the likelihood of early detection of cancers. While this approach may be clinically appropriate, the genetic testing of children raises many ethical and philosophical concerns that are the focus of intense scrutiny.

Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome (BWS) occurs with a frequency of 1 in 13,700 births. BWS is associated with a wide spectrum of phenotypic stigmata, including hemihypertrophy/hyperplasia, exomphalos, macroglossia, gigantism, and ear pits (posterior aspect of the pinna). Laboratory findings may include profound neonatal hypoglycemia (extremely common), polycythemia, hypocalcemia, hypertriglyceridemia, hypercholesterolemia, and high serum α-fetoprotein levels.197 With increasing age, phenotypic and laboratory features of BWS become less pronounced. While neurocognitive defects are not universal in BWS, early diagnosis of the condition is crucial to avoid deleterious neurological effects of neonatal hypoglycemia and to initiate an appropriate screening protocol for tumor development.198 The increased risk for tumor formation in Beckwith-Wiedemann syndrome (BWS) patients is estimated at 7.5% and is further increased to 10% if hemihyperplasia is present. Tumors occurring with the highest frequency include Wilms tumor, hepatoblastoma, neuroblastoma, rhabdomyosarcoma, and ACC.2,199

The genetic basis of BWS is complex. Various 11p15 chromosomal or molecular alterations have been associated with the BWS phenotype and its tumors.200 Abnormalities in this region impact an imprinted domain, indicating that it is more likely that normal gene regulation in this part of chromosome 11p15 occurs in a regional manner and may depend on various interdependent factors or genes. These include the paternally expressed genes IGF2 and KCNQ10T1 and the maternally expressed genes H19, CDKN1C, and KCNQ1.201 Detectable chromosomal abnormalities associated with BWS are extremely rare, with only 20 cases having been associated with 11p15 translocations or inversions. However, in each of these cases, the chromosomal break point is always found on the maternally derived chromosome 11.202 This parent-of-origin dependence in BWS suggests that the chromosome translocations disrupt imprinting of a gene in the 11p15 region. On the other hand, BWS-associated 11p15 duplications are always paternally derived, and the duplication break points are heterogeneous.203 Paternal uniparental disomy, in which two alleles are inherited from one parent (the father), has been reported in approximately 15% of sporadic BWS patients.204 It is interesting that the insulin/IGF2 region is always represented in the uniparental disomy, although the extent of chromosomal involvement is highly variable. Alterations in allele-specific DNA methylation of IGF2 and H19 reflect this paternal imprinting phenomenon.204 A minority of BWS patients exhibit demonstrable constitutional DNA sequence alterations, the most common being CDKN1C mutations.205 Twenty-five percent to 50% of BWS patients exhibit biallelic rather than monoallelic expression of IGF2. Another 50% have epigenetic mutations resulting in loss of imprinting of KCNQ10T0. Of interest, epigenetic changes, such as methylation and chromatin modification, occur in many pediatric and adult cancers,206 indicating the value of the BWS model in understanding the broad scope of molecular changes in cancer (eTable 443.1).

Despite the associated cytogenetic and molecular findings for some patients, no single diagnostic test exists for BWS. Nevertheless, the often characteristic phenotype should alert family practitioners and pediatricians to the possibility of the diagnosis and the need to institute an intense clinical surveillance program for early cancer detection. These include regular (every 3 months) abdominal ultrasound, focusing particularly on the kidneys, liver, and suprarenal areas, and serum alpha-fetoprotein for the detection of hepatoblastoma. This program is considered standard until approximately 10 years of age. Treatment is directed at the particular tumors.

Gorlin Syndrome

Nevoid basal cell carcinoma syndrome, or Gorlin syndrome, is a rare autosomal dominant disorder characterized by multiple basal cell carcinomas; developmental defects, including bifid ribs and other spine and rib abnormalities; palmar and plantar pits; odontogenic keratocysts; and generalized overgrowth.207 The sonic hedgehog (SHH) signaling pathway directs embryonic development of a spectrum of organisms. Gorlin syndrome appears to be caused by germ-line mutations of the tumor suppressor gene PTCH, a receptor for SHH.208,209 Medulloblastoma develops in approximately 5% of patients with Gorlin syndrome. Furthermore, approximately 10% of patients diagnosed with medulloblastoma by the age of 2 are found to have other phenotypic features consistent with Gorlin syndrome and also harbor germ-line PTCH mutations.210 Although Gorlin syndrome develops in individuals with germ-line mutations of PTCH, a subset of children with medulloblastoma harbor germ-line mutations of another gene, SUFU, in the SHH pathway, with accompanying LOH in the tumors.211 Of further note, mice with heterozygous PTC deletions develop RMS.212 Although RMS is not associated with Gorlin syndrome, the mouse studies suggest a possible link between PTC signaling and RMS.213

Multiple Endocrine Neoplasia

The multiple endocrine neoplasia (MEN) disorders comprise at least three different diseases: MEN type 1 (MEN1), MEN type 2A (MEN2A), and MEN type 2B (MEN2B), which are all cancer predisposition syndromes that affect different endocrine organs. The most common features of MEN1 are parathyroid adenomas (about 90% of the cases), pancreatic islet cell tumors (50–75% of the cases), and pituitary adenomas (25–65% of the cases).214MEN2A is associated with medullary thyroid carcinoma, parathyroid adenoma, and pheochromocytoma. MEN2B is a related disorder but with onset of the tumors in early infancy, ganglioneurinoma of the gastrointestinal tract, and skeletal abnormalities.

While MEN1 is caused by mutation in the tumor-suppressor gene, MEN1, MEN2A, and MEN2B are caused by mutations in the proto-oncogene RET. Genetic testing is possible by direct mutation analysis of the 10 exons of the MEN1. Linkage analysis mapped the gene for MEN2A to chromosome 10q11.2,215,216 a region where the proto-oncogene RET, known to be translocated in thyroid papillary carcinoma, is located. Further studies confirmed constitutional mutations in the RET proto-oncogene in families with MEN2A and MEN2B; in addition, a wider range of somatic mutations was found in patients with nonhereditary medullary thyroid carcinoma.217 The pattern of mutations seen in MEN2 families does not follow the two-hit-hypothesis for tumor suppressor genes: the RET proto-oncogene is not inactivated, and there is no loss of the second allele in the tumors. Thus, the predisposition to cancer in families with MEN2 is based on the inheritance of an activating mutation in the RET proto-oncogene.

The fact that by the age of 15 years, 28% of mutation carriers show clinical or biochemical evidence of disease218 justifies the early start of screening for documented MEN1 families. The screening is very complex and should at least include the determination of serum ionized calcium and prolactin levels, but measurements of pituitary, parathyroid, and pancreatic hormones may be added. Guidelines for managing these patients and families have been published.218 In light of the clear guidelines and an effective clinical screening regimen, genetic testing should be offered to families and patients to avoid unnecessary investigations and to focus resources on individuals at risk. As in MEN1, the biochemical screening in MEN2 is very complex and genetic testing should be offered to avoid unnecessary screening. Because the risk of developing medullary thyroid carcinoma is virtually 100% in mutation carriers, all children with a confirmed mutation should receive a prophylactic thyroidectomy by the age of 5 years (MEN 2A) or 1 year (MEN 2B), respectively.219 In addition, mutation carriers require lifelong screening for pheochromocytoma and parathyroid disease.

Continued refinement of molecular analysis for tumor diagnosis, prognosis, and development of novel therapeutic avenues for children with cancer should profoundly influence disease outcomes. The use of molecular screening as a tool for identifying children at risk for the purpose of developing rational clinical surveillance guidelines is more controversial. Several issues are worth noting. Based on recommendations of the American Society of Clinical Oncology,220 genetic testing should be undertaken only with fully informed consent, including elements of risk assessment, psychological implications of test results (both benefits and risks), risks of employer or insurance discrimination, confidentiality issues and options, and limitations of medical surveillance or prevention strategies. While screening for some mutations, such as RET or RB1, are associated with clearly defined beneficial medical management decisions that lead to improved outcomes, presymptomatic identification of other gene mutations, such as in LFS, are of less obvious clinical benefit. Regardless of the scenario, the complexity of the issues underlies the importance that these patients and families be referred to an experienced multidisciplinary team, including oncologists, geneticists, psychologists, and genetic counselors to facilitate the most appropriate management.