Part 3. Solid Tumors

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents.Osteosarcoma’s destruction of normal bone architecture and its production of malignant osteoid can permit its identification from skeletal remains, allowing the recognition that it is an ancient disease. Among the earliest malignant bone tumor described in humans, it was presumed to be identified in the humerus of a male Celt from 800 to 600 BC.1 A probable osteosarcoma dates back to 250 AD in the pelvis of a young individual from Ancient Egypt. A definite osteosarcoma with a classic sunburst appearance was found in the remains of the femur of a Peruvian from approximately 1100 to 1200 AD.1 The term osteosarcoma was introduced by Alexis Boyer (1757–1833), and the first large clinical-pathological description of sarcomas of bone was performed by Samuel Weissel Gross (1837–1889).2 In this description, the tendency for hematogenous and not lymphatic dissemination, as well as the occurrence of skip metastases, led to the suggestion that amputation be performed at a distance beyond the primary lesion.2

The diagnosis of osteosarcoma can be made based on histologic examination of pathologic material, staging can be performed with imaging procedures, and multimodality therapy including chemotherapy and surgery successfully treat the majority of patients. Much information has been acquired about the natural history and biology of this disease. Despite all of these advances, osteosarcoma remains enigmatic. The biology of the tumor is incompletely understood, and recent trials modifying therapy have failed to produce further advances. Patients with localized disease have a 5-year survival of at least 70%; patients with metastatic or recurrent disease have a < 20% chance of long-term survival despite aggressive therapies. These figures have changed little in the past 2 decades.3-6 A challenge over the next few years will be to develop strategies to understand better the biology and behavior of this tumor, to facilitate further advances in its treatment. The objective of this chapter is to review what is known about osteosarcoma, including its epidemiology, etiology, biology, presentation, evaluation, staging, pathology, treatment, and future approaches. This will establish a context in which emerging strategies are being developed which may change the outlook for patients with this disease.

Epidemiology/Etiology

Several features characterize the epidemiology of osteosarcoma and have been taken as providing some clues as to its pathogenesis. Osteosarcoma has a bimodal age distribution with the first peak in the second decade of life and the second among older adults. The approximate incidence of osteosarcoma is 4.8 per million children younger than 20 years of age.7 There is some variability in the annual incidence rate as shown in eFigure 453.1. The incidence by age is as low as 0.3 per million in children under the age of 5 years, 2.8 per million between the ages of 5 and 9 years, 8.3 per million between the ages of 10 and 14 years, and 9.4 per million between the ages of 15 and 19 years.7 The incidence by age at diagnosis is shown in eFigure 453.2. There are approximately 400 new cases per year in those under 20 years of age in the United States.8 Primary bone tumors account for approximately 5% to 10% of all new pediatric cancer diagnoses in the United States, of which osteosarcoma is the most common.7 The male-to-female ratio is approximately 1.3:1 in this age range. The incidence in males is 5.2 per million as compared to 4.5 per million in females. It is slightly more common in black children than in white children with an incidence of 5.2 per million in black individuals under 20 years of age compared to 4.6 per million in whites.7

The peak age of incidence coincides with a period of rapid bone growth in young people, which suggests a correlation between bone growth and the evolution of osteosarcoma.8,9 Other evidence supporting this relationship includes the higher incidence of osteosarcoma in large dog breeds as compared with small breeds10,11 and the earlier peak age in girls as compared with boys, corresponding to the earlier age of their growth spurt.12 At the same time, it must be recognized that osteosarcoma arises in many patients both well before and long after the adolescent growth spurt.

Radiation exposure is a well-documented epidemiologic factor, but as the interval between irradiation and the appearance of osteosarcoma is typically long, this is not relevant to most pediatric patients.5,8,13-18 The incidence of osteosarcoma is dramatically increased among survivors of retinoblastoma. In the hereditary form of this disorder, germline mutations of the retinoblastoma gene are common.19,20 This is the probable basis of the increased frequency of secondary cancers in this population, because the rate in survivors of unilateral sporadic retinoblastoma is much less.21,22 Germline mutations in the p53 gene can lead to a high risk of developing malignancies including osteosarcoma, which has been described as the Li-Fraumeni syndrome.23,24 Patients with Rothmund-Thomson syndrome, particularly those with a RecQL4 mutation, are at high risk of developing osteosarcoma.12,25 Patients with Werner and Bloom syndrome are at a high risk of developing osteosarcoma among other cancer predispositions.26-28 Other predisposing factors include a history of disorders of bone metabolism (ie, Paget disease of the bone and fibrous dysplasia).29-31

A viral etiology has been suggested based on evidence that bone sarcomas can be induced in select animals by viruses, in hamsters by the injection of cell-free extracts of human osteosarcoma, and the presence of SV40-like DNA sequences in up to 50% of human osteosarcomas in one study.32,33 The presence of the sequences suggested that contamination of poliovirus by SV40 which occurred in North America between 1955 and 1963 and further spontaneous transmission of the virus may be an etiologic factor in osteosarcoma.34,35 The most definitive study demonstrating SV40 is not a major epidemiologic factor in the development of osteosarcoma was a negative serologic study for antibodies in a large cohort of osteosarcoma patients.34,35 Trauma has often been associated with the diagnosis of osteosarcoma, but little evidence exists to support a causal relationship.8

Osteosarcoma at the molecular level is an extremely complex tumor.36-39 Although many of the alterations present in the tumors have been cataloged, the molecular complexity precludes placing the tumor’s pathogenesis into a conceptual framework.5,12 Osteosarcoma is defined by its production of disorganized osteoid.40-42 Producing bony matrix is a cellular behavior or a phenotype, not a genetic marker. Considerable variability exists in the predominant matrix produced, described as the histologic subtype, but the presence of even a small area of osteoid in association with a malignant spindle cell is viewed by pathologists as sufficient to make the diagnosis. Cytogenetics, specific molecular markers, and immunohistochemistry are not typically used to assist in making the diagnosis.41 Despite this phenotypic definition, the clinical behavior of high-grade osteosarcoma is remarkably homogeneous. Histologic subtype does not influence the chemotherapy utilized or the tendency to metastasize early in its natural history or to a great extent chemotherapy response or prognosis.8,40,41 Given this phenotypic definition, it is perhaps not surprising that at the molecular level considerable variability exists, making understanding of the fundamental biology of this tumor difficult.

All sarcomas can be broadly characterized into two groups: those that are genetically complex and those that have relatively simple karyotypes in association with a recurrent chromosomal translocation.43,44 Osteosarcoma is a prototypical member of the former, and larger, group of sarcomas. As is characteristic of these group of sarcomas, p53 is frequently altered but not prognostic.45 The tumor’s development is associated with a variety of etiologic factors, and each tumor has a large number of genetic alterations, many of which are recurrent across different tumors but none of which is universally present.36-39

Unlike the majority of tumors that arise in adults which are predominantly of epithelial origin, osteosarcoma does not have an obvious multistep progression. Low-grade osteosarcomas cannot be identified as a precursor lesion in the majority of children and adolescents who develop a high-grade osteosarcoma.43,45 The equivalent of a premalignant dysplastic lesion or a carcinoma in situ is not known to exist for osteosarcoma, similar to most pediatric malignancies. In defining the molecular pathogenesis of osteosarcoma, the first lesion that can typically be analyzed is already a fully malignant cancer. This fact coupled with molecular complexity makes it extremely difficult to define the molecular features essential to the tumor’s pathogenesis. Despite these difficulties, a considerable amount is known about specific molecular features that may be associated with osteosarcoma development which will be discussed further.

Genetic Alterations Involved in Osteosarcoma Pathogenesis

Despite its genetic complexity, numerous clues exist as to processes and genetic pathways that may be associated with the pathogenesis of osteosarcoma. These clues are derived from mouse models of osteosarcoma, human predisposition syndromes, etiologic-environmental factors, and studies of genetic alterations in tumors.5,12 In contrast to most cancers, too many models rather than too few produce osteosarcoma. Given the rarity of osteosarcoma, many of these genetic events may not be clinically relevant or, alternatively, the development of osteosarcoma is restricted by constraints that are poorly understood which may be temporally or immune related as examples.26 Models and analyses that provide clues as to the pathogenesis of osteosarcoma are summarized in eTable 453.1.

Perhaps the most compelling data potentially defining the pathogenesis of osteosarcoma are humans with germ-line genetic alterations that lead to an osteosarcoma predisposition. The syndromes that predispose to osteosarcoma are summarized in eTable 453.2. In individuals with hereditary retinoblastoma that is associated with a germ-line mutation in the Rb gene, secondary malignancies are common, and 40% of these malignancies will be osteosarcoma.21,22,46,47 Both osteosarcoma and retinoblastoma occurring in patients with hereditary retinoblastoma have somatic loss of the normal Rb allele and resultant complete absence of a functional Rb protein.20,48,49 Support for the importance of Rb gene involvement in osteosarcoma derives from the frequent derangement of this pathway in tumor specimens.20,48-50 On the other hand, the vast majority of Rb gene abnormalities are inherited in an autosomal dominant manner and have high penetrance.51 Hence, the lack of a history of prior retinoblastoma in the vast majority of patients who are diagnosed with sporadic osteosarcoma, suggests germ-line alterations of Rb in this patient population is not common. Low penetrance Rb mutations have been reported, but their incidence among patients with sporadic osteosarcoma is not known.51 Similarly in Li-Fraumeni syndrome with germ-line alterations in p53, malignancy is frequent.24,52,53 Approximately 10% of these are osteosarcoma which is the second most common tumor they develop.23,24,52,53 In a manner analogous to the Rb gene, the p53 pathway is frequently deranged in tumors.54,55 Among patients with sporadic osteosarcoma, only 3% were found to harbor unsuspected alterations in germ line p53.56,57

The data supporting RECQ DNA helicases involvement in the pathogenesis of osteosarcoma are somewhat more limited. RECQ DNA helicases are conserved proteins that separate the complementary strands of DNA duplexes. The three RECQ DNA helicases involved in cancer predisposition syndromes are RECQL4 in Rothmund-Thomson syndrome, RECQL2 in Werner syndrome, and RECQL3 in Bloom syndrome. In the context of Rothmund-Thomson syndrome, mutation of RecQL4 is associated with osteosarcoma development.25 In sporadic osteosarcomas RecQL4 is rarely altered, suggesting it does not play a role in the pathogenesis of these tumors.58 In contrast to Rothmund-Thomson syndrome, patients who have a 30% incidence of osteosarcoma and less than 10% of patients with either Werner syndrome or Bloom syndrome develop osteosarcoma.27,28

Although genetic alterations in humans which are associated with osteosarcoma have a more directly established relevance, murine models offer additional evidence as to genetic alterations and exposures that can predispose to osteosarcoma. A large number of murine models develop osteosarcoma. Transgenic mice engineered to overexpress SV40 develop osteosarcoma, with the clinical pattern dependent upon the promoter driving expression.59 This is supportive of the hypothesis that the p53 and Rb tumor suppressor genes have a role in osteosarcoma development as the SV40 large T antigen abrogates the function of both of these pathways.59 Knockout mice with mutant or absent p53 develop osteosarcoma along with a variety of other tumors. Depending upon the particular p53 alteration, osteosarcoma develops in between 5% and 50% of the mice.60-63 Knocking out the Rb gene is lethal at embryonic stages in mice; thus, it is not possible to assess whether or not these mice are prone to developing osteosarcoma.64 More recent studies have used conditional knockouts of p53 and Rb to demonstrate that these models develop osteosarcoma.26,65 Transgenic overexpression of several oncogenes, including myc and fos, can produce osteosarcoma.66,67 Almost 100% of transgenic mice that ubiquitously overexpress fos develop osteosarcoma.66 Transgenic mice that conditionally overexpress myc in fibroblasts/osteoblasts develop osteosarcoma.67 Irradiating mice leads to the development of osteosarcoma, as does chronic exposure to parathyroid hormone.68,69

A number of epidemiologic factors are associated with the development of osteosarcoma. Although growth is not a genetic event, this has provided support that the growth hormone-insulin like growth factor axis is involved in osteosarcoma pathogenesis. Multiple studies have linked periods of rapid growth to the development of osteosarcoma, with the incidence for this disease peaking during the second decade of life. Osteosarcoma has a predilection for the rapidly growing long bones of the extremities, arising in the metaphysis of the bones the site of new bone formation by the growth plates.5,6,8,12 Other factors suggesting an association with growth include increased incidence in taller individuals, in the majority of reported studies, as well as its earlier peak incidence in females concordant with their earlier growth spurt.31,70-73 Perhaps among the more frequently cited data in making this assertion is the high incidence of osteosarcoma among large breed dogs and its almost complete absence among smaller dogs.74-76 More recent studies have suggested that the incidence of osteosarcoma does not vary within canine breeds, suggesting the determining factor may be their underlying genetics rather than height as a phenotype.11,77 More recent explanations of canine breed height variability based on a finite number of polymorphisms in the insulin-like growth factor axis may give further credence to that view.78 Independent of growth, accelerated bone turnover likely plays a role in the etiology of osteosarcoma, given its association with disorders of bone metabolism (ie, Paget disease of the bone and fibrous dysplasia).29,30,79 Another predisposing factor includes exposure to nonspecific DNA damaging agents such as ionizing radiation.21,47,80-83 The mechanism by which these etiologic factors predispose to this specific malignancy remains unclear.

Multiple molecular alterations resulting in inactivation of tumor suppressor genes and overexpression of oncogenes are observed with considerable variability in every osteosarcoma. With the multitude of genetic lesions in osteosarcoma, it is difficult to discern which of these events is fundamental in tumorigenesis.12 The most consistent feature across human predisposition syndromes, murine predisposition syndromes, and analyses of human tumors are alterations of the Rb and p53 tumor suppressor pathways.

The inactivation of these pathways is accomplished through a variety of genetic alterations. These alterations typically occur in a nonoverlapping manner so that only one mechanism of Rb inactivation and one mechanism of p53 inactivation are present in an osteosarcoma. Genetic lesions in the Rb gene, mapped to 13q14, have been shown to be present in approximately 70% of primary osteosarcoma tumor samples.20,48-50 Approximately 60% of osteosarcoma tumors will have loss of heterozygosity at 13q, the site of Rb. Gross structural rearrangements of the Rb gene are present in approximately 30% of osteosarcoma tumors.20,48-50,84,85 Cyclin-dependent kinase-4 (CDK4) in complex with cyclin D1 (CCND1) is responsible for the phosphorylation of Rb; therefore, amplification or overexpression of these genes results in functional inactivation of the Rb signaling pathway. Amplification of the 12q13-15 chromosomal region that contains CDK4 or the gene is observed in approximately 5% to 10% of osteosarcomas.86 The INK4A gene, localized to 9p21, encodes p16INK4A, a tumor suppressor that inhibits the CDK4-CCND1 complex, thereby halting cell cycle transition. Alterations of INK4A result in an Rb pathway disruption. Deletions in the INK4A region are observed in approximately 5% to 10% of osteosarcomas.86 Deletions of INK4A, by virtue of an alternative reading frame that encodes another tumor suppressor, p19INK4A or p14ARF, functionally inactivates p53 as well.87 Analogous to Rb, a significant proportion of sporadic osteosarcoma samples have some molecular modification abrogating normal p53 function. In osteosarcoma tumor samples, alterations in p53 consist of point mutations (occurs in 20% to 30%), gross gene rearrangements (occurs in 10% to 20%), and loss of one 17q allele (occurs in 75% to 80%).54-56,88,89 Alterations in other genes encoding proteins that regulate p53 have also been implicated in the pathogenesis of osteosarcoma. MDM2, mapped to chromosome 12q13-14, which negatively modulates p53 function by binding the protein, concealing its activation site, and facilitating its proteasomal degradation, is amplified in about 10% of osteosarcomas.54 Another negative regulator of p53 is COPS3 (constitutive photomorphogenic homolog subunit 3) that is amplified in about 25% of cases of osteosarcoma.90 COPS3 amplification, INK4A deletion, MDM2 amplification, and TP53 mutations seem to be mutually exclusive, again suggesting that any of these alterations in the TP53 tumor suppressor pathway is sufficient in regard to the contribution of this pathway to tumorigenesis in osteosarcoma.90 These molecular alterations provide a loss of cell cycle checkpoint function and normal DNA damage response which are essential steps in malignant transformation.91-93 The Rb gene plays an essential role in cell cycle regulation, and the p53 gene product plays a major role in the cellular response to DNA damage, a more full description of which is beyond the scope of this chapter.94-97 The invariable presence of alterations of Rb and p53 suggests that inactivation of these pathways may be essential for osteosarcoma’s development, but this does not necessarily establish these events as early steps in tumor pathogenesis, as other events may drive the development of these abnormalities and serve as the initiating genetic event.

Cell of Origin

Tumors may be defined by both the cells they originate from and the genetic events that lead to tumor formation. Osteosarcoma has traditionally been believed to arise from an osteoblast, but the data supporting that statement are rather limited.40,41 Several lines of evidence suggest that osteosarcoma has a more pluripotent potential and may in fact arise from a more primitive precursor.26,65,98 Normal osteoblasts derive from a pluripotent mesenchymal stem cell that can differentiate into cartilage, muscle, stroma, fat, and fibrous tissue in accordance with well-defined differentiation pathways not unlike hematopoiesis. The presence of osteoid has led to the traditional viewpoint that osteosarcoma is derived from osteoblasts. It is well known that great variability exists in the histological patterns seen in this tumor and in the degree of osteoid production, so that extensive review of the pathologic material may be required to demonstrate tumor osteoid. It is also known that these tumors are capable of differentiating toward fibrous tissue, cartilage, or bone with resulting chondroblastic, fibroblastic, and osteoblastic components suggesting the cell of origin may be more pluripotent than an osteoblast. Tumors with various patterns of differentiation are traditionally referred to as histologic subtypes. It is well established that many tumors have mixed patterns.40,41 It is also known that the histologic subtype does not impact in a major way on chemotherapy response or outcome, and the patients are treated identically irrespective of subtype, suggesting these various patterns of differentiation are reflective of a single clinical entity. At present, the factors that control histologic appearance in osteosarcoma are poorly understood.40,41 Osteosarcoma could arise from a mesenchymal stem cell that acquires patterns of osteoblastic differentiation during transformation. Similarly, osteosarcoma may arise from an osteoblast and the pluripotent capacity can be acquired through dedifferentiation during the transformation process. Both the acquisition and loss of differentiation properties can be observed in the potentially analogous development of hematopoietic malignancies.26,65 Several recent findings in murine models have also implicated mesenchymal stem cells as a possible progenitor; however, all of this data are far from definitive.99 Even if one accepts osteoblasts as the cell of origin of osteosarcoma, these cells exist in various pools and at various stages of maturity. These pools include the bone marrow, growth plates, and the periosteum; it is unclear which pool serves as the usual cell of origin.

Redundancy

In osteosarcoma, numerous studies have been undertaken cataloguing the genetic abnormalities present in tumor samples. Many of these will be discussed in the subsequent sections in which they are considered in the context of functional roles. In addition to the known alterations, osteosarcomas have tremendous chromosomal complexity with numerous whole chromosome alterations as well as a large number of regions with consistent genetic gains and losses.36,37,39,100 These sites have been characterized to a variable extent, and many of the genes altered in osteosarcoma have not been identified or characterized.79,101,102 Before going through these alterations, it may be worthwhile to mention that investigators have demonstrated that only a few genetic elements are necessary to transform a normal cell into a cancer as only a few fundamental processes need to be deranged.103 To become malignant, a normal cell needs a signal to progress through the cell cycle, loss of checkpoint control, telomere length stabilization, and loss of contact inhibition/dependence.103 Osteosarcoma clearly does not need all of the alterations it possesses to achieve these tasks, and many genetic events are likely to be bystander effects related perhaps to genetic instability. This is perhaps most evident in expression of growth factor receptors in osteosarcoma. Osteosarcoma has been reported to express IGF, VEGF, HER2, ErbB-4, PTHR, and HGF.104-122 Many, if not all of these, pathways are redundant. In osteosarcoma numerous genetic abnormalities are present, each of which individually could lead to the development of a malignancy. This redundancy makes it difficult to decipher the relative timing and importance of each of these events. Quite similar to the molecular abnormalities, the epidemiology suggests multiple etiologic factors may play a role in osteosarcoma development. Some of these factors may have differing levels of importance in different patient populations with the overall contribution of each of these factors unknown.

Growth Factors/Signal Transduction Pathways

Growth factor receptors and autocrine or paracrine stimulation may contribute to abnormal cell proliferation in the pathogenesis of osteosarcoma. These feedback loops lead to loss of external regulation of cell proliferation, motility, and angiogenesis. The previously described epidemiologic association with growth suggests that these pathways may play an important role in the initiation or potentially the pathogenesis of osteosarcoma.

Insulin-like growth factor-1 (IGF-1), a major regulator of skeletal growth, has been found to be mitogenic in in vitro models of osteosarcoma.114,123-126 Survival of osteosarcoma cell lines in vitro has been shown to be dependent on exogenous IGF-1, and proliferation of these cell lines can be inhibited by blocking signaling through the IGF-1 receptor via monoclonal antibodies or antisense oligonucleotides.104,124,125 Insulin-like growth factor receptor-1 (IGF-1R) has been found to be abundantly expressed in osteosarcoma cells.104 IGF-1R has emerged as the predominant receptor in cancer biology which contributes not only to cellular proliferation, but perhaps more importantly to malignant transformation, and protection from apoptosis.127 Overexpression of IGF-1 and its receptor may therefore play a role in the pathogenesis of osteosarcoma by contributing to the oncogenic transformation and providing a proliferative and survival advantage to malignant cells.127 Of the growth factor circuits involved in the pathogenesis of osteosarcoma, the contribution of IGF-1/IGF-1R to the malignant potential of this tumor has been identified to be of major importance.128

Overexpression of the c-met proto-oncogene tyrosine kinase receptor (Met) and its ligand, hepatocyte growth factor (HGF) or scatter factor, in osteosarcoma cell lines suggests a role for the Met in the metastatic phenotype of osteosarcoma. Binding of HGF or scatter factor to the Met/HGF receptor stimulates both cell proliferation and motility, features associated with the malignant potential of tumor cells.129,130 The predominant expression of the Met/HGF receptor on epithelial cells, along with its ligand being produced primarily by cells of mesenchymal origin, suggests a paracrine or autocrine signaling system in governing stromal-epithelial interactions.130-132 In a study of 17 osteosarcoma tumor samples, 60% expressed the HGF receptor at high levels.130 When primary and metastatic tumors from the same patient are compared, metastatic tumors have higher Met expression than the primary tumors, again suggesting its role in potentiating the metastatic phenotype of osteosarcoma.128,131,132

Platelet-derived growth factor (PDGF) is another potent mitogen for cells of mesenchymal origin. Coexpression of PDGF and its receptors has been observed in various human solid tumors and correlates with inferior prognosis and metastasis in some, suggesting an autocrine or paracrine mechanism driving cellular proliferation and differentiation, chemotaxis, and survival in these tumors.133-141 The role of PDGF/PDGF-R in osteosarcoma has been explored.142 In one immunohistochemical study, PDGF-AA and its receptor, PDGFR-alpha, was found in 34% and 27% of osteosarcoma tumor samples, respectively, with coexpression of both proteins common. Another analysis demonstrated PDGF A and PDGFR-alpha expression in 80% of osteosarcoma tumor samples, with coexpression found in 76% of the patients; the expression of either growth factor or receptor, or both, correlated significantly with a shorter event-free survival.142

Other tyrosine kinase receptors have been implicated in the pathogenesis of osteosarcoma. The c-erbB-2 proto-oncogene (also called HER2/neu), located on 17q12, encodes a protein structurally homologous to the epidermal growth factor receptor, although its actual ligand has not yet been identified. The importance of expression of ErbB family of tyrosine kinase receptors for tumor growth or clinical outcome has been demonstrated in a variety of human cancers, most notably in invasive breast cancers and non-small-cell lung cancer. In these carcinomas, activation of intracellular mitogenic signal transduction pathways is implicated in contributing to tumorigenesis. The data in osteosarcoma are somewhat controversial, with some investigators reporting overexpression, associated with either favorable or unfavorable prognosis, and other investigators reporting lack of overexpression of erb-B2.107,108,111,116,143-153 In a study of 26 osteosarcoma tumor samples, 42% expressed c-erbB2; the expression of erb-B2 in this cohort was associated with a poor prognosis.108 Conversely, another study found HER2 overexpression associated with increased survival and less metastases.143,144 A more recent report utilizing immunohistochemistry and tissue microarray data failed to find significant overexpression of the HER2 protein or gene amplification at the c-erb-B2 locus.152,153 Various explanations may account for the discrepancies between studies, including methodology and interpretation of immunohistochemical staining, antibody clones utilized, and cross-reactivity with other membrane antigens, along with inherent biologic variability among osteosarcoma samples tested in various institutions representing different geographical areas. ErbB-4 is another member of the epidermal growth factor receptor family that has been implicated in the pathogenesis of osteosarcoma. Hughes et al. evaluated the expression pattern of erbB family members in primary osteosarcoma tumor samples and found cell surface expression of EGFR and erb-B2, along with nuclear expression of the p80 isoform of erb-B4.110 In a follow-up study, the authors demonstrated constitutive phosphorylation of this erbB family of receptor tyrosine kinases in osteosarcoma cells and suggested their role in tumorigenesis.111

Identification of growth factors that may play a role in the pathogenesis of osteosarcoma leads one to consider the downstream signal transduction pathways resulting from activation of their receptors. The majority of signaling by the aforementioned receptors is transduced by 3 interrelated parallel pathways; Ras/Raf/mitogen-activated protein kinases (MAPK), phosphatidylinositol 3’-kinase/AKT (PI3K/AKT), and mammalian target of rapamycin (mTOR).154-157 Each of these pathways is composed of a series of kinases where signal is transmitted through phosphorylation of amino acids, tyrosine, serine, or threonine. Transmission of signal in this manner permits it to occur rapidly as no RNA or protein synthesis is required. This also permits marked signal amplification.

The MAPK pathways play pivotal roles in cell proliferation, differentiation, and survival. The closely related MAPK pathways are regulated through a series of phosphorylation steps in a 3-component module. This feature ensures the tight control of the signals transduced by MAPK pathways in terms of specificity, intensity, and duration.158,159 MAPK is among the most common dysregulated pathways identified in tumors, as indicated by activating mutations frequently occurring in RAS or BRAF in human cancer, particularly in colon cancer and malignant melanoma.160 Some evidence suggests that this pathway may mediate differentiation in osteoblasts.158,161 To date, little information is available regarding the role of this pathway in osteosarcoma. Several sources of data suggest this pathway may be constitutively active in osteosarcoma.142

The PI3K/AKT and mTOR signal transduction pathways play an important role in transducing a variety of receptor signals. mTOR perhaps most importantly serves as the intracellular pathway through which IGF signaling is transduced.156,162 Signal transduction via PDGF receptors occurs predominantly through the PI3K/AKT pathway.142 Ezrin mediates its growth and survival signal via both PI3K/AKT and mTOR but not MAPK.163 These signaling pathways have a variety of components and downstream targets, a discussion of which is beyond the scope of this chapter. In the context of osteosarcoma, little is known about these pathways, although several publications demonstrate activation of these pathways with a variety of ligands and with associated biological affects. Analogous to the MAPK pathway, both PI3K/AKT and mTOR have been suggested to be involved in the differentiation of mesenchymal stem cells, in this case toward the adipogenic lineage.164 The mTOR pathway plays a major role in cell growth and differentiation in a variety of tumors, including osteosarcoma. PI3K/AKT has been implicated in osteosarcoma’s metastatic process as well as chemotherapy sensitivity.165,166

Other Oncogenes

Many other cellular proto-oncogenes are altered in osteosarcoma and have been suggested as contributing to its molecular pathogenesis. The MYC proto-oncogene, localized to 8q24, functions in the regulation of cell proliferation, cell growth, inhibition of differentiation, and apoptosis. It has been found to be overexpressed in a subset of osteosarcoma tumor samples.167-170 The MYC gene is amplified in 44% of osteosarcomas.168-170 c-Myc expression has been suggested to be related to metastasis.167 As mentioned previously, certain myc overexpressing transgenic mice have an osteosarcoma predisposition.67 Fos was initially identified as being related to osteosarcoma through recognition that mice inoculated with FBJ murine virus developed these tumors.171,172 The viral protein that induces osteosarcoma is v-fos, which led to the identification of the structurally related c-fos protein as an oncogene.172-174 Fos heterodimerizes with Jun to regulate the AP1 transcription factor. The AP1 transcription factor is known to be regulated by vitamin D, transforming growth factor beta, and parathyroid hormone—all known to be involved in the regulation of bone growth, indicating a possible basis for its association with osteosarcoma development. Overexpression of Fos oncoprotein is found in the majority of osteosarcomas and is potentially associated with metastasis or recurrence.167,175,176

Genetic Complexity

In addition to the known loss of tumor suppressor genes and gain of oncogenes, osteosarcoma has tremendous chromosomal complexity with numerous other genes affected by gross genetic changes. As one example of the genetic complexity, spectral karyotyping reveals an average of 39 chromosomal rearrangements per tumor.177 Some of the genetic alterations appear to be random, but recurrent regions of chromosomal gain and loss are present.38,178,179 In many of these cases, the target of the genetic amplification and loss are known, and in many cases they are not.79,102 The most common genetic losses and regions with losses of heterozygosity involve chromosomes 2, 3, 6, 9, 10, 13, 17, and 18 with the Rb gene located on chromosome 13 and p53 located on chromosome 17.38 Loss of heterozygosity on chromosome 18 is also seen in Paget disease and chromosome 3 in Brachmann-de Lange syndrome, a bone dysmorphology syndrome.29,101,102 The most common regions of amplification include regions within chromosomes 1, 5, 6, 8, 12, 16, and 17, with MYC located on chromosome 8 and MDM2 on chromosome 12. Candidate genes for some of the amplified regions include PRDM16, CDC5L, NFKBIE, IFNG, MGRN1, PMP22, MYCD, TOP3A, MAPK7, and COPS3.38

Immortalization

Immortalization heralded by telomere length stabilization has been explored as contributing to the pathogenesis of osteosarcoma. Telomeres cap chromosome ends and serve to potentiate the replicative process in cells by compensating for the loss of genetic material secondary to inability to replicate end DNA.180,181 Maintaining telomere length is regarded as a fundamental necessity for overcoming cellular senescence in tumor cells. Maintaining telomere length is accomplished by most cancers via activation of an enzyme, telomerase (TERT).103 TERT activity is only present in a minority of osteosarcomas and does not seem to be essential to tumorigenesis. When present in osteosarcoma tumor samples, TERT activity has been shown to be low in comparison to positive controls.182 The clinicopathological correlation of telomerase has varied: an inverse relationship was reported between TERT activity and pulmonary metastases, and another study found decreased survival associated with TERT expression in primary osteosarcoma tumor samples. Alternative mechanisms of telomere lengthening (ALT), a recombination-based method, is the mechanism of maintaining telomere length seen more often in osteosarcoma. About 60% of osteosarcoma tumor samples utilize ALT. The aggressiveness of osteosarcoma has been associated with telomere integrity: a subset of osteosarcoma patients who lacked both TERT activity and evidence of ALT demonstrated a more favorable prognosis.183-185

Bone Differentiation

The complex molecular alterations in osteosarcoma may not only involve dysregulation of cellular pathways involved in proliferation and immortalization, but also disruptions in differentiation. Osteosarcoma can be regarded as potentially evolving from a primitive pluripotent progenitor cell that retains its proliferative capacity while undergoing partial differentiation, or alternatively, a more differentiated precursor cell that dedifferentiates and gains an ability to proliferate. The differentiation status of osteosarcoma tumors is variable. Transforming growth factor-beta (TGF-β) isoforms play an important role in regulating bone formation.186-188 An immunohistochemical analysis demonstrated expression of one or more TGF-β isoforms, particularly TGF-β3, in all of the 25 high-grade osteosarcoma tumor samples studied.189 TGF-β3 was correlated with disease progression. Another study demonstrated increased expression of TGF-β2 and VEGF correlated with osteosarcoma grade.112 Bone morphogenetic proteins (BMPs), part of the TGF-β superfamily, are multifunctional cytokines that regulate bone and skeletal development. BMPs are involved in the differentiation of mesenchymal cells to cells of osteoblastic lineage, and in the differentiation of immature osteoblasts into mature osteoblasts.190,191 In vitro data suggest that BMPs stimulate growth of osteosarcoma cells.191 Numerous BMPs and their receptors are highly expressed in osteosarcoma tumors, implicating their importance in autocrine or paracrine growth stimulation of osteosarcoma.190 Overexpression of BMP Receptor II has been associated with poor prognosis and metastatic potential in osteosarcoma.190 BMPs have been demonstrated to induce the expression of c-fos MRNA in animal models of osteoblast formation, suggesting that the sustained expression of c-fos has a role in the pathogenesis of osteosarcoma.191

The process of osteoblast differentiation is driven by wingless (Wnt) signaling within the mesenchymal stem cell. Wnts, encompassing the wingless and int genes, encode a family of highly conserved, secreted ligands that play an important role in osteoblast development and tumorigenesis.192 Wnt binds to the cell surface receptor frizzled (Frz) and the coreceptor lipoprotein–related proteins 5 and 6 (LRP-5/6), leading to the stabilization and accumulation of β-catenin that in turn elicits a variety of effects including induction of differentiation and proliferation.192-194 Aberrant activation of Wnt signaling is associated with many human cancers.195 Cytoplasmic or nuclear accumulation of β-catenin has been found in a majority of osteosarcomas and has correlated with its metastatic potential.196 Expression of LRP5, seen in 50% of osteosarcomas, is associated with a less-differentiated phenotype and metastasis.193 Overexpression of dickkopf-3 (Dkk-3), a Wnt agonist, and inhibition of LRP5 by using a dominant-negative LRP5 receptor in osteosarcoma cell lines was shown to inhibit tumor cell motility and invasion.192 The Wnt signaling pathway, along with other signaling pathways that are involved in osteoblast differentiation, can be induced by parathyroid hormone. PTH plays a central role in the regulation of bone and mineral metabolism and has been implicated in the pathogenesis of osteosarcoma.120 In response to PTHrP, osteoclastic resorption of bone is stimulated, and as a result, growth factors such as transforming growth factor-β (TGF-β) and IGF-1 are released from the surrounding extracellular matrix, further stimulating PTHrP secretion and additional tumor growth.120 This positive-feedback loop is believed to be involved in bone metastasis.197

Metastasis

Metastasis is a complicated process that involves motility, migration, degradation of extracellular matrix, extravasation, survival during transit through vasculature, invasion, and growth.198 Given the complicated nature of the process, many factors are likely to play a role in metastases. Several of the factors that have been more extensively studied in the context of osteosarcoma will be discussed.

Chemokine stromal cell-derived factor 1 (SDF-1) is a cytokine-like protein expressed on the surface of vascular endothelial cells. Through binding to its chemokine receptor, CXCR4, it plays a role in cytoskeleton rearrangement, adhesion to endothelial cells, and chemotaxis.199 CXCR4 mRNA has been reported to be expressed in osteosarcoma and associated with the presence of metastases at the time of diagnosis. These results have not been consistently supported in other reported work.115,200-204 In vitro assays have shown that migration of osteosarcoma cells expressing CXCR4 follows an SDF-1 gradient and that their adhesion to endothelial and bone marrow stromal cells is promoted by SDF-1 treatment.202 This study also provided a rationale for the propensity of osteosarcoma to metastasize to the lung, where SDF-1 concentration is high. Studies have demonstrated prevention of pulmonary metastasis in a murine model by the administration of a CXCR4 inhibitor, suggesting molecular strategies inhibiting this axis as a therapeutic target.205

Ezrin is a membrane-cytoskeleton linker protein that allows direct cellular interactions with the microenvironment, facilitating signal transduction through growth factor receptors and adhesion molecules, thereby regulating cell migration and metastasis among other processes. In an orthotopic model of murine osteosarcoma, ezrin expression was 3-fold higher in the more aggressive K7M2 cell line which correlated with its metastatic potential when compared to the less aggressive K12 cell line.206 In follow-up experiments, ezrin expression was found to provide an early survival advantage for pulmonary metastatic osteosarcoma, which is in part mediated by AKT.163 A significant correlation between high ezrin expression and poor outcome in osteosarcoma has been shown, with shorter DFS and higher risk of metastatic relapse, supporting animal model data.206

Other factors that likely play a role in osteosarcoma metastases are expression of matrix metalloproteinases and other degradative enzymes. The expression of these enzymes is necessary in order to degrade the extracellular matrix, permitting extravasation into the vasculature.207-210 Loss of expression of Fas appears to be necessary for survival of metastatic osteosarcoma cells in the lung environment, because lung tissue highly expresses Fas ligand, and binding of Fas to Fas ligand activates apoptotic pathways in the malignant cell.211-214

Angiogenesis

In addition to cell proliferation and motility, another feature contributing to the behavior of tumors is the ability to induce the proliferation and migration of endothelial cells to allow the formation of new capillaries. Vascular endothelial growth factor (VEGF) is a peptide that acts as a mitogen for endothelial cells, directing new vessel formation.105,109,112,115,215 The role of angiogenesis in the pathogenesis of osteosarcoma has been explored via expression analysis of VEGF in tumor samples. In one such study, VEGF expression was seen by immunostaining in 62% of 17 primary osteosarcoma tumor samples; VEGF positivity correlated with poor prognosis and a higher metastatic rate.215 In another study, the presence of VEGF expression in tumor cells following neoadjuvant chemotherapy was associated with a poor outcome.105,109,215

Drug Resistance

The efficacy of chemotherapy for the treatment of osteosarcoma can potentially be hindered by the presence of drug resistance. Possible mechanisms of drug resistance in osteosarcoma include alterations in a variety of proteins related to drug transport, drug efflux, drug metabolism, target interaction, and downstream response.12 Of the potential mechanisms of resistance, P-glycoprotein expression has been the most extensively studied in osteosarcoma. P-glycoprotein is a transmembrane ATP-dependent efflux pump protein encoded by the multidrug resistance (MDR1) gene, which is responsible for the efflux of numerous chemotherapeutic agents, including doxorubicin.107,216-228 Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) quantification studies have explored P-glycoprotein expression in osteosarcoma and demonstrated overexpression of P-glycoprotein in 23% to 45%, with decreased survival reported for patients with P-GP-positive tumors.107,216-228 A meta-analysis also proposes that P-glycoprotein is associated with an increased risk of disease progression.221 Though the literature suggests that P-glycoprotein may be a marker of drug resistance and aggressiveness in osteosarcoma, numerous studies contradict this finding, and a prospective clinicopathological national intergroup study concluded that there was no correlation between P-glycoprotein expression and percentage of osteosarcoma tumor necrosis after induction chemotherapy or event-free survival in localized osteosarcoma.228 Overexpression of MDR1, the gene encoding P-glycoprotein, also has been explored in regard to its role in the progression of osteosarcoma and its prognostic relevance.226,227,229 Though a small pilot study showed a trend toward a worse outcome in patients exhibiting high levels of MDR1 expression, the larger, prospective investigation did not delineate any correlation between MDR1 expression and disease progression in patients with osteosarcoma, with patients with either very low or very high levels of MDR1 fairing poorly.227 The value of P-glycoprotein and MDR1 expression as predictors of prognosis remains extremely controversial.217,225,228,230

High-dose methotrexate with leucovorin rescue is a major component of current protocols for the treatment of osteosarcoma. High-dose methotrexate is much more effective than conventional-dose methotrexate in the treatment of osteosarcoma—a finding that is not observed in other malignancies, implying a mechanism of intrinsic methotrexate resistance within osteosarcoma tumor cells.231-235 Methotrexate is a potent inhibitor of dihydrofolate reductase, a key enzyme for intracellular folate metabolism. In experimental systems, resistance to methotrexate can occur through a variety of mechanisms, including impaired transmembrane transport of the drug via the reduced folate carrier, upregulation of dihydrofolate reductase, and diminished intracellular retention secondary to polyglutamylation.236,237 Studies have demonstrated that impairment of methotrexate influx and upregulation of dihydrofolate reductase occur in osteosarcoma.231-235,238,239 The alterations in drug influx are mediated by changes in reduced folate carrier expression and sequence.231-235 Some studies have linked alterations conferring methotrexate resistance to clinical parameters.231-235 These studies have not been large enough to permit making any definitive conclusions.

Clinical Presentation

The most common features in the clinical presentation of osteosarcoma are pain and swelling in the involved region, with or without an associated soft tissue mass. Pain is often attributed to trauma or vigorous physical exercise, both of which are common in the patient population.3-6,8,240,241 In adolescents, pain is at times attributed to more common benign conditions such as growing pains. Symptoms are usually present for several months before the diagnosis is made. The median time from onset of symptoms to diagnosis is 4 months.242-246 Occasionally, patients present more dramatically with a pathologic fracture. The most common primary site is the metaphysis of a long bone, but osteosarcoma can occur in any bone of the body. Approximately half of all osteosarcomas originate in the region around the knee.40,41 The most common sites of disease are the distal femur, the proximal tibia, and the proximal humerus. Cases occurring in the long bones outnumber those in the axial skeleton.40,41 The anatomic distribution of osteosarcoma is shown in Figure 453-1.7 Approximately 20% of patients present with radiographically detectable metastatic disease. Eighty percent of patients present with localized disease, but the vast majority of this group are known to have subclinical, microscopic metastases.8 The most frequent site of metastases is the lung. Much less frequently, metastases at initial diagnosis can occur in other bones. The vast majority of patients at presentation do not have systemic symptoms such as fever or weight loss even with overt metastatic disease. Death from osteosarcoma is almost always the result of progressive pulmonary metastasis.8 Bone lesions, although extremely painful, are usually not life threatening.3-6,8,240 Pain at multiple sites does not rule out the possibility of osteosarcoma, as this can result from metastatic disease. Metastases in the lungs produce respiratory symptoms only with extensive involvement. When osteosarcoma is widely metastatic, more frequently at recurrence than at the time of initial diagnosis, it can spread to the central nervous system or gastrointestinal tract.

Evaluation

The evaluation of suspected osteosarcoma begins with history, physical examination, laboratory evaluations, and plain radiographs. History may be significant only for pain. Physical examination is typically remarkable only for a soft tissue mass, not always present, at the primary site.3-6,8,240,241 Laboratory evaluation is seldom revealing. Elevation of the serum alkaline phosphatase and lactate dehydrogenase is observed in a large proportion of patients but is not sensitive or specific.8 The differential diagnosis for pain at a bone site in this age group includes the malignant bone tumors, osteosarcoma, and Ewing sarcoma family tumors along with a variety of benign bone tumors, as well as a variety of nonneoplastic conditions, with osteomyelitis among the more common. In a patient with a suspected bone tumor, obtaining a plain radiograph of the site is critical for making an appropriate diagnosis.3-6,8,240,241

The diagnosis of osteosarcoma is typically suspected from its radiographic appearance. Osteosarcoma can present as a lytic, a sclerotic, or a mixed lytic-sclerotic lesion. Typically, patients with extremity osteosarcoma have a poorly defined, lytic and sclerotic lesion involving the metaphyseal portion of the bone. As with other malignant tumors in bone, plain films reveal bone destruction with poorly defined zones of transition. Ossification in the soft tissue suggestive of bone spicule formation in a “sunburst”’ pattern is classic for osteosarcoma but is not a sensitive or specific feature.40,41 Cortical destruction with periosteal new bone formation can lead to the appearance of a Codman’s triangle. In addition to the lesion’s appearance, features such as location in the metaphysis and the bone involved as well as the patient’s age help to distinguish osteosarcoma from other clinical entities.40,41 Appearance of the tumor on plain radiographs can be highly suggestive of the diagnosis. In addition, plain radiographs can identify pathologic fractures that may be present at diagnosis. A typical radiograph of a patient with an osteosarcoma in the femur is shown in Figure 453-2. Despite the utility of plain radiographs, computerized tomography (CT) and magnetic resonance imaging (MRI) both offer improved resolution and better delineation of tumor extension and are routinely performed with suspected osteosarcoma.247-249 The cross-sectional imaging of the primary tumor should include the entire bone from which the tumor arises and the closest adjacent joint in order to properly screen for skip metastases.247-250 In most institutions, MRI is performed as the initial cross-sectional imaging. A typical cross-sectional image is shown in eFigure 453.3. It is worth noting that in some studies directly comparing the imaging modalities for patients with osteosarcoma, a statistically significant difference between CT scans and MRIs in their ability to predict the involvement of bone, muscle, joints, and the neurovascular bundle was not found.249 These studies suggest that both CT and MRI have advantages and disadvantages and either but not both should be used for initial imaging.249

The diagnosis of osteosarcoma can be accurately predicted in two thirds of cases based on radiographic location and appearance but should never be made from images alone.41 It is mandatory to obtain a biopsy for pathological confirmation of the diagnosis. The biopsy must be performed by someone experienced in performing these procedures as a poorly placed biopsy can necessitate a subsequent amputation or have other morbidities.251-256 Cross-sectional imaging should be preformed prior to the biopsy, as the tumor’s appearance can be altered by the biopsy itself. The diagnosis of osteosarcoma is made by histologic examination of the biopsy.40,41 The histopathologic appearance of osteosarcoma will be described subsequently.

Staging

Prior to initiation of therapy, accurate staging is necessary to determine the extent of disease which has prognostic significance.257 Routine posteroanterior and lateral radiographs of the chest allow detection of metastases in the majority of cases. CT of the chest is more sensitive in detecting pulmonary metastases and has become the imaging procedure of choice.258 The appearance of pulmonary metastases in osteosarcoma is shown in eFigure 453.4. However, there are false-positive results, particularly with smaller lesions.259 In patients presenting with bone tumors that are highly suggestive of a malignant tumor, it is recommended that the chest radiographs and the chest CT be obtained prior to the biopsy.8,260 Children frequently receive general anesthesia for the biopsy which can induce atelectasis, making interpretation of the imaging more difficult if obtained subsequently. Careful radiographic imaging of the lungs is critical at diagnosis as the presence of lung metastases influences both the patient’s treatment and the patient’s anticipated prognosis.240,241,245,261,262

A radionuclide bone scan, with methylene diphosphonate labeled with technetium-99m is also routinely performed at diagnosis.258,263 The typical appearance is shown in eFigure 453.5. It defines the extent of the primary tumor and is useful in the detection of “skip lesions” within the same bone, as well as distant bone metastases.264-267 A skip lesion is shown in eFigure 453.6. An unusual presentation of osteosarcoma is multifocal osteosarcoma, a rare entity in which multiple synchronous skeletal tumors are present at diagnosis with each lesion resembling the primary tumor radiographically, suggesting a multicentric origin. It is unclear whether such sarcomas arise in multiple sites or if one of the lesions is the true primary that has metastasized rapidly to other skeletal sites in the absence of lung metastases.268-270 Although positron emission tomography (PET) is being used with increasing frequency in sarcomas (especially Ewing sarcoma family tumors and rhabdomyosarcoma), PET scans are not yet routinely performed in osteosarcoma patients. There are a few studies that show PET scans may be useful in predicting response to chemotherapy, and differentiating postoperative changes from recurrent tumor.271-275 However, its usefulness in osteosarcoma has not yet been validated.

Although not specifically for the purposes of staging, the initial evaluation includes a determination of the patient’s baseline organ function as a prerequisite for receiving chemotherapy.3,4,6,8,9,240,241,276 Successful treatment of high-grade osteosarcoma requires the use of systemic chemotherapy. Many of the agents have both acute and long-term toxicity; therefore, it is essential to obtain baseline studies prior to the initiation of chemotherapy so that these toxicities can be monitored. Baseline evaluations of renal function, cardiac function, and hearing are usually performed. All young men past the age of puberty typically are offered the opportunity to carry out sperm banking. Newer techniques for maintaining fertility of women have been developed, but their indications have not completely been established.3,4,6,8,9,240,241,276

The most widely utilized staging system for bone tumors including osteosarcoma is the system developed by Enneking and associates.277 This system categorizes localized malignant bone tumors by grade (low grade—stage I or high grade—stage II) and by the local anatomic extent (intracompartmental—A or extracompartmental—B). The compartmental status is determined by whether or not the tumor extends through the cortex. Patients with distant metastases are stage III.277 There are very few high-grade intramedullary lesions (ie, stage IIA), because most high-grade osteosarcomas break through the cortex early in their natural history. In the younger age groups, the vast majority of osteosarcomas are high-grade lesions; hence, virtually all patients are stage IIB or III distinguished by the presence or absence of detectable metastatic disease.3,6,8

Prognostic Factors

The outcome of osteosarcoma patients depends on several factors, including how it is treated. The most consistent prognostic factor at diagnosis is the presence of clinically detectable metastatic disease, which confers an unfavorable prognosis.241,245,261,262,278,279 Among patients presenting with localized disease, few factors are sufficiently predictive so as to allow stratification of therapy or more precise determination of a patient’s likelihood of survival.280 The site of the primary tumor has some prognostic significance, with axial lesions prognostically inferior to tumors of the appendicular skeleton.245 Both serum lactate dehydrogenase and alkaline phosphatase have been suggested to correlate with outcome; higher levels of either enzyme predict an inferior prognosis.281-284 These factors most likely influence outcome by reflecting the size of the tumor or its resectability. One predictor of outcome which has been used to stratify therapy is the percentage of tumor necrosis (ie, “Huvos” grade) assessed at the time of definitive resection after a period of preoperative chemotherapy.285,286 A tumor demonstrating complete necrosis and one demonstrating residual viable tumor are shown in eFigure 453.7. Although tumor necrosis based on Huvos grading has been shown to correlate with disease-free survival, this variable cannot be assessed at diagnosis and thus cannot serve as a true prognostic factor because stratification of therapy based on Huvos grade occurs after 10 weeks of chemotherapy/surgery. Thus far, modifying therapy after necrosis assessment has not altered outcome.287-293 This may be because tumor necrosis in response to preoperative chemotherapy may reflect innate tumor biology rather than chemotherapy effectiveness, because clinical trials that augmented therapy based on poor histological response to induction chemotherapy have not yet improved overall survival for patients.294 However, as will be discussed subsequently, the idea of stratifying therapy based on response to preoperative chemotherapy is still being tested in the current international cooperative group clinical trial for osteosarcoma. In some studies, patient age and directly measured tumor size have been suggested to be prognostic.41,245,278-280 In patients with disease that is metastatic at diagnosis, the number of pulmonary nodules as well as their laterality are of prognostic significance.262,295-297

Pathology

The diagnosis of osteosarcoma is based on histopathologic criteria and depends on the identification of malignant spindle cells associated with the production of tumor osteoid. The diagnosis of osteosarcoma is made based on its histologic appearance using routine light microscopy.40-42 Typically, molecular analyses do not assist in making the diagnosis. Great variability exists in the histologic patterns seen in this tumor and in the degree of osteoid production, so that extensive review of the pathologic material may be required to demonstrate tumor osteoid.40-42 Osteosarcomas are capable of differentiating toward fibrous tissue, cartilage, or bone, and can have chondroblastic, fibroblastic, and osteoblastic components. Chondrosarcomas and fibrosarcomas are distinguished from osteosarcoma, with which they can be histologically confused, by their lack of production of osteoid.40-42

The World Health Organization classifies osteosarcomas into central (medullary) and surface (peripheral) tumors and recognizes a number of subtypes within each group.298 Conventional high-grade central osteosarcoma accounts for 80% to 90% of all osteosarcomas, and it is the predominant form in children and adolescents. It is a primary intramedullary high-grade malignant tumor in which the neoplastic cells produce osteoid, even if only in small amounts. Based on the predominant differentiation of the tumor cells, the most frequent subtypes of conventional osteosarcoma are osteoblastic (50%), chondroblastic (25%), and fibroblastic (25%).40-42 In osteoblastic osteosarcoma, the tumor appears to be a mixture of large, atypical, spindle-shaped cells that are cytologically highly malignant, with large irregular nuclei and abnormal mitotic figures. Interspersed are areas of osteoid production and calcification in close proximity to the malignant cells (eFig. 453.8). The value of these subclassifications (osteoblastic, chondroblastic, fibroblastic) is unclear, as they are very dependent on sampling with mixed histology frequent in a given tumor.40-42 Moreover, no difference in clinical behavior or outcome based on subtype has been consistently recognized.289,299-301

Conventional high-grade osteosarcomas may develop on the surface of bone and are similar to conventional osteosarcoma both histologically and clinically. Telangiectatic osteosarcoma is an unusual subtype that is notable because it appears to be a purely lytic lesion on plain radiographs, leading to diagnostic confusion with aneurysmal bone cysts and giant cell tumors. These tumors are grossly cystic and histologically demonstrate dilated blood-filled spaces with viable tumor cells typically confined to the periphery of the lesion.40-42 Although they were initially reported to be associated with an unfavorable outcome, subsequent studies treating them as a typical high-grade osteosarcoma fail to demonstrate any difference in prognosis with some suggesting they are more responsive to chemotherapy.289,299-301 Small-cell osteosarcoma is a subtype notable because it can be easily confused histologically with Ewing sarcoma.302-304 Some clinicians treat small-cell osteosarcoma as a Ewing sarcoma rather than with typical osteosarcoma chemotherapy. Other rarer subtypes and clinical variants such as the malignant fibrous histiocytoma subtype of osteosarcoma, osteosarcoma of the jaw, giant cell rich, extraosseous, and juxtacortical (parosteal) osteosarcoma occur primarily in older patients and therefore will not be discussed further. Similarly, low-grade lesions rarely occur in pediatric patients and also will not be discussed.40-42

Treatment

Chemotherapy for Localized Disease

Although approximately 20% of all newly diagnosed patients present with overt metastatic disease, all patients are assumed to have micrometastatic disease at presentation based on the historical data that 75% to 90% of patients with what was considered “nonmetastatic” osteosarcoma based on radiographic studies treated with radical surgery develop distant metastatic disease within 3 to 6 months following resection alone.305-309

In the 1970s, several investigators reported that chemotherapy had activity against relapsed or metastatic osteosarcoma. The cytotoxic agents with the highest single-agent activity in osteosarcoma include methotrexate (at high doses only, 30% to 40%), cisplatin (~30%), doxorubicin (30% to 40%), and ifosfamide (~30%).3 When these agents were administered to patients with relapsed or metastatic osteosarcoma, tumor responses were observed.310-318 It must be noted that compared to other pediatric malignancies, the list of drugs shown to be effective was relatively short. Many studies with single-agent therapy were discouraging: agents such as vincristine and 5-fluorouracil were not shown to be active in osteosarcoma.241,319-324 Based on the activity in metastatic and recurrent disease, it was hypothesized that in the absence of any clinically detectable or bulk disease, chemotherapy may be even more effective and may result in improved long-term disease-free survival. With this rationale, several investigators embarked on trials of adjuvant chemotherapy for the treatment of localized osteosarcoma. In the 1970s and early 1980s, all of these trials were single arm, nonrandomized treatments employing adjuvant chemotherapy, comparing results against historical controls. Every reported trial of adjuvant chemotherapy for osteosarcoma demonstrated a disease-free survival superior to the historical rate of 25%.241,310,312,325-331

Not all investigators were convinced that chemotherapy was appropriate for all patients with osteosarcoma as the use of historical comparisons can be flawed. Other changes in the management of osteosarcoma had occurred during the period of time the early chemotherapy studies were being performed, such as the routine use of CT to assess for pulmonary metastases at the time of diagnosis. As trials of chemotherapy were limited to those without clinically detectable metastases, it was postulated the improved survival might be the result of identification of a cohort of patients with an intrinsically better outcome. Earlier diagnosis or improved surgical technique could have also contributed to improved outcome.241,332-335 In the early 1980s, investigators at the Mayo Clinic carried out the first randomized trial of adjuvant chemotherapy for osteosarcoma. All patients had surgical resection of the primary tumor and had no evidence of clinically detectable metastatic disease. Patients were randomly assigned to observation or to adjuvant chemotherapy. There was no difference between the two groups and the disease-free survival for each was 40%. The disease-free survival for patients treated with surgery alone was better than any previously reported trial, and there was no apparent advantage to the use of adjuvant chemotherapy. This suggested that the natural history of the disease had changed, and this could have accounted for the improved outcomes observed in the adjuvant chemotherapy trials.335

To clarify the issues surrounding the value of adjuvant chemotherapy, the Multi-Institution Osteosarcoma Study (MIOS) was initiated. Patients were eligible for study entry after amputation or tumor resection if they did not have clinically detectable metastatic disease. The intent of the study was to randomize patients between observation and adjuvant multiagent chemotherapy. Many patients enrolled in the study were not randomized, with most of those being treated with chemotherapy. Results were analyzed for both the randomized patients and the larger group of patients who were not randomized with both having identical results. Patients who did not receive chemotherapy after surgery had a probability of disease-free survival of 11% as compared to 66% for those who received chemotherapy. This study unequivocally demonstrated that the natural history of osteosarcoma had not changed, and adjuvant chemotherapy was of clear benefit.336 With longer follow-up, an overall survival advantage with adjuvant chemotherapy became apparent in agreement with the disease-free survival rates observed earlier.337 The majority of patients surviving 3 years without evidence of recurrence were likely cured.337 Since this study, systemic chemotherapy has been the standard of care for all patients with high-grade osteosarcoma.241

In the same era, Rosen and colleagues introduced the concept in osteosarcoma of giving chemotherapy before carrying out definitive surgery on the primary tumor.285,338 This treatment was initially referred to as neoadjuvant or preoperative chemotherapy but recently has been referred to as induction chemotherapy. The concept of induction chemotherapy arose from the need for time to make the custom endoprosthesis required for a limb salvage procedure, creating an interval during which chemotherapy could be administered. In addition, theoretical advantages of induction chemotherapy included early treatment of micrometastatic disease and facilitation of the eventual surgical procedure, because of tumor shrinkage and decreased vascularity. It also became possible to examine the histologic response of the tumor to induction chemotherapy to assess the effectiveness of therapy.41,241,285,286,338 A strong correlation between the degree of necrosis (Huvos grade) and the probability of subsequent disease-free survival was observed, and this prognostic value has been confirmed in multiple clinical trials.278,338-344 A theoretical concern with this approach is that the delay in removal of the bulk tumor could lead to the emergence of chemotherapy resistance. A prospective trial by the Pediatric Oncology Group addressed this concern by randomizing patients to immediate definitive surgery or to receive induction chemotherapy followed by definitive surgery with the total chemotherapy given in both arms identical.345 Reports from this study indicate that there was no statistically significant advantage to immediate surgery.345 Given the advantages in facilitating limb salvage procedures and in assessing chemotherapeutic efficacy, the use of induction chemotherapy became the standard treatment approach for osteosarcoma. Numerous studies have demonstrated that resection of all sites of bulk tumor is similarly necessary for a favorable outcome; additional surgery is discussed subsequently. One can speculate that this is because the sites of macroscopic disease contain the largest reservoir of tumor cells with the potential to contribute not only to metastasis but also to the development of resistance.4 In fact, one can argue that up-front surgery with the availability of modular prosthetics should be explored further.4,241

Soon after the identification of the prognostic value of the degree of necrosis following induction chemotherapy, it was suggested that chemotherapy be modified for the patients with less necrosis (currently referred to as either standard or poor responders, and variably defined as < 90% or < 98% tumor necrosis) in order to increase their probability of disease-free survival. Modifying therapy to achieve a better outcome in patients with a poor histologic response was initially reported as showing benefit in the results of the T10 protocol at Memorial Sloan-Kettering Cancer Center.338 Longer follow-up of this patient population, however, showed no benefit to the therapy intensification.261,262,278 Numerous cooperative groups and institutions, including the Children’s Cancer Group, the Cooperative Osteosarcoma Study Group (COSS), and Instituto Rizzoli, among others have undertaken studies using a similar strategy, delivering a variety of intensified regimens to patients with standard response in an attempt to improve their outcome. The vast majority of studies have demonstrated that intensified therapy cannot change the outcome of a patient who has had a standard response.288,293,340,343,344,346-349 Intensification of therapy in the induction period to increase the proportion of patients with greater amounts of necrosis (currently referred to as favorable responders) likewise did not change the outcome.241,287

Several trials have been conducted to clarify the role of the various chemotherapeutic agents in the treatment of osteosarcoma as well as their method of delivery. The combination of bleomycin, cyclophosphamide, and actinomycin D was once common in osteosarcoma treatment, but subsequent studies demonstrated the combination was not effective and it is no longer recommended.350 Studies of presurgical chemotherapy administered via the intra-arterial route have been performed. Most commonly, doxorubicin and cisplatin were administered directly into the arterial supply of extremity tumors with the theoretical advantage that the drug delivery to the tumor vasculature and tumor would be maximized. High local drug concentrations were documented by pharmacokinetic studies, and dramatic responses in primary tumors were observed.351-357 Reviewing studies of intra-arterial chemotherapy in general demonstrate that if improvements occur in local control, which is controversial, it may be at the expense of treating systemic disease. An update of studies from the MD Anderson Hospital where intra-arterial chemotherapy was pioneered indicates that the overall disease-free survival for patients treated with intra-arterial cisplatin, definitive surgery, and postoperative adjuvant chemotherapy is 60%, a somewhat disappointing result for a single institutional trial.358 The use of intra-arterial chemotherapy has declined in recent years.241

The last North American–based randomized Phase III pediatric cooperative group study (INT-0133), jointly administered by the Children’s Cancer Group and the Pediatric Oncology Group, addressed two study questions.359 The study tested whether the addition of ifosfamide or muramyl tripeptide–phosphatidyl ethanolamine (MTP-PE) to the three other agents used in the standard treatment of osteosarcoma (doxorubicin, cisplatin, high-dose methotrexate) would improve the probability of disease-free survival.359 The schema for that study is shown in eFigure 453.9 Muramyl tripeptide is a component of the bacillus Calmette-Guerin (BCG) cell wall. It is conjugated to phosphatidyl ethanolamine and liposome encapsulated to improve delivery to the reticuloendothelial system.360-362 The primary rationale supporting the use of this relatively nonspecific immune adjuvant were the encouraging results obtained in a prospective randomized trial of this compound in canines.363 The results of this trial did not demonstrate a benefit in either event-free survival or survival for the addition of ifosfamide.359,364 The addition of muramyl tripeptide–phosphatidyl ethanolaminein the initial report suggested it did not provide an advantage for patient event-free survival.359 A subsequent report demonstrated an overall survival advantagefor the addition of muramyl tripeptide–phosphatidyl ethanolamine.364 The overall survival at 6 years for the patients who did not receive the muramyl tripeptide–phosphatidyl ethanolamine was 70% and for those who did was 78% as shown in Figure 453-3.364 These overall survival numbers represent among the best reported in a cooperative group study for the treatment of this disease, and the 3-drug chemotherapy backbone remains the standard of care for the treatment of osteosarcoma. The importance of MTP-PE in the treatment of osteosarcoma remains under intensive discussion.364-366

The Children’s Oncology Group has completed a series of pilot feasibility studies assessing increased-dose doxorubicin with dexrazoxane cardioprotection in combination with the other standard agents, increased-dose ifosfamide and etoposide in combination with the other standard agents, or standard-dose ifosfamide in combination with increased-dose doxorubicin with dexrazoxane cardioprotection in patients with a standard chemotherapy response. The results of this study have been presented but not published as of yet, but the treatments were generally tolerable.241

The Scandinavian Sarcoma Group (SSG) performed 4 nonrandomized clinical trials for high-grade osteosarcoma localized to the extremities.293,342,367 The first study (SSG-II: 1982–1989) was based on the MSKCC T10 regimen. All patients received 4 courses of high-dose methotrexate preoperatively. Good responders continued with methotrexate, BCD (bleomycin, cyclophosphamide, and dactinomycin), and poor responders were treated with the addition of cisplatin.367 The subsequent SSG osteosarcoma trial (SSG-VIII: 1990–1997) used a 3-drug combination of methotrexate, doxorubicin, and cisplatin (MAP) preoperatively. Good responders were given 3 further cycles of MAP, and poor responders received 5 cycles of etoposide and ifosfamide. Both studies failed to improve the outcome in poor histological responders.342 The following study, a joint Italian/Scandinavian study (ISG/SSG I: 1997–2000), explored the benefit of adding high-dose ifosfamide (15 g/m2) to induction therapy, which also did not improve outcome compared to previous trials.293 In the next SSG osteosarcoma study, SSG XIV, all patients received 2 MAP cycles preoperatively and 3 further cycles postoperatively. Poor responders also received ifosfamide (10 to 14 g/m2). The results of this study have not been published.241,368 At the same time, the German-Austrian-Swiss Cooperative Osteosarcoma Study Group (COSS) performed a series of studies using multiagent chemotherapy.343,344 COSS 80 (1979–1982) compared the effect of cisplatin to that of the BCD combination. Patients were randomized to receive either drug within a course of sequential multidrug chemotherapy with doxorubicin and high-dose methotrexate. After surgery, patients were further randomized to receive or not to receive interferon in addition to chemotherapy. There was no difference in disease-free survival rates in the patients receiving BCD versus cisplatin or receiving interferon versus no interferon.344 The COSS 82 study tried to spare some patients from the chronic toxicity of doxorubicin and cisplatin by using these agents only in poor responders to a less-aggressive preoperative regimen. Poor responders were not salvaged by postoperative intensification, suggesting that osteosarcoma chemotherapy should be as aggressive as possible from the time of its initiation.343 The European Osteosarcoma Intergroup (EOI) used shorter dose-intense regimens in a series of large prospective randomized controlled studies.369-371 An initial study demonstrated no benefit to the addition of methotrexate to doxorubicin and cisplatin, but the regimen produced outcomes considerably inferior to those reported by other groups.369 A second study compared doxorubicin and cisplatin alone with a multiagent combination similar to the T10 regimen. This study reported no benefit from the longer multiagent protocol, although the overall 5-year event survival for the whole study group was once again inferior to most reported results.370,371

The EURAMOS 1 clinical trial, launched in 2005, is a large ongoing phase III trial. The EURAMOS study is a collaboration between the Children’s Oncology Group, COSS, EOI, and the SSG.241,368 The aim of the study is to evaluate whether it is possible to improve the outcome of both poor and good responders to preoperative chemotherapy by modification of postoperative chemotherapy. All patients receive preoperative methotrexate, doxorubicin, and cisplatin (MAP) as given in the control arm of the INT-0133 study. Poor responders are randomized to receive MAP with or without the addition of high-dose ifosfamide and etoposide. Good responders will continue on MAP chemotherapy and are randomized to the addition of pegylated interferon-α.241,368

Radiation Therapy

Radiation therapy has not been widely utilized in the upfront treatment of osteosarcoma as studies in the prechemotherapy era did not show benefit when compared to surgery alone.372-376 Also, conventional teaching states that osteosarcoma is not sensitive to radiotherapy at doses that would allow the safe administration of systemic combination chemotherapy. Most studies show that dosages in excess of 50 Gy are typically required for tumor sterilization.372-376 At these doses, complications are common, and risks of radiation therapy usually outweigh any potential benefits. However, with advances in techniques such as intensity-modulated radiation therapy, several small series have reported excellent symptom and tumor control in patients with large unresectable (or incompletely resected) tumors, or in patients who refused surgery.377-380 Additionally, several recent studies reported clinical improvement in the palliative setting.377-380 Thus, radiation therapy is becoming a potential option for patients whose primary tumors are in sites where appropriate surgery is impossible and for palliation.241 Efficacy of radiation in unresected tumors and in up-front therapy remains to be defined.241 Two reports describe the use of radiation therapy following incomplete surgery with positive margins.380,381 These reports suggest that external beam radiation therapy reduced the risk of local recurrence for patients with incomplete resection compared to historical controls.380,382,383 In addition to traditional external beam irradiation, radiopharmaceuticals have also been employed in the treatment of osteosarcoma. The most widely studied is samarium (Sm)-153 ethylene diamine tetramethylene phosphonate (153Sm-EDTMP).384,385 This compound has been shown to have high bone specificity and may be effectively used in the palliative setting.241

Surgery

Preparation for surgical management should begin even before the initial biopsy is performed.6,241 Careful review of all pertinent imaging and laboratory studies is imperative. An appreciation of tumor size, extension, location, joint involvement, proximity to neurovascular structures, and necrosis are important factors that need to be carefully weighed if a representative biopsy sample and successful surgical plan are to be realized.251-256 Poorly planned biopsies may compromise a patient’s oncologic and functional outcome, because the complete biopsy tract and overlying skin need to be resected when the definitive resection is performed.251-256 In general, a biopsy should be obtained by the same surgeon who will ultimately perform the subsequent resection.241 Whether the biopsy is performed as an open or closed surgical procedure is controversial. A closed procedure may be feasible on an outpatient basis and not require general anesthetic in selected children. An open procedure obtains more tissue, facilitating diagnosis and potentially permitting the conduct of biological studies.6,251-256

Tumors at the primary site as well as at all sites of radiographically visible metastatic disease require local control.241 Osteosarcoma is considered resistant to radiation therapy; therefore, local control is usually surgery. Advances made in surgery have significantly improved the clinical practice and options available. Complete surgical resection of primary and metastatic disease is the cornerstone for successful treatment. Surgical management of osteosarcoma involves both the complete extirpation of the tumor and its ensuing bone and soft-tissue reconstruction.6,241 Historically, most patients were treated with an amputation, but advances over the past 3 decades have made limb-salvage surgery a viable option, with similar survival outcomes when properly performed.386-388 Complete surgical resection takes precedence and should not be compromised for a preferred functional or reconstructive outcome.241 Although limb-salvage surgery is a popular choice among patients and their families, this may not be realistic in every case.6,241

Surgical treatment of extremity osteosarcoma should be by definition a wide or radical excision. This means that the tumor, its adjacent reactive zone, and a normal cuff of tissue in all planes should be resected. Wide excisions can be achieved through a variety of means, the most radical being an amputation or a disarticulation, which is essentially an amputation through a joint.241 In contrast, limb-salvage surgery spares much of the normal tissue and calls for reconstruction of the surgical defect with biologic materials, synthetic materials, or a combination of the two.6,241 Because osteosarcoma most commonly affects the metaphyseal region, the surgical resection is often intra-articular or through the joint. If there is extension into the joint or across the joint, an extra-articular resection may be necessary.241 For intra-articular resections, reconstruction may be achieved with osteoarticular allograft obtained from a bone bank and size matched to the patient’s anatomy. Cadaveric osteoarticular grafts restore bone stock, offer biologic ingrowth, and spare both the joint surface and the growth plate of the adjacent bone. This bone will often heal at the host–graft junction, but it never fully revascularizes or remodels and is therefore prone to fracture, infection, and nonunion.241 It is also expected to undergo arthritic changes at an accelerated rate compared with viable bone and cartilage and in time often requires joint resurfacing.6,241

Endoprosthetic joint replacements are metal implants that historically were custom built for individual patients.241 Today, endoprosthetic systems are modular and allow for customization to be achieved in the operating room by mixing and combining standard components. These systems in certain cases can even be utilized to replace an entire long bone along with both adjacent joints.6,241 Metal endoprosthetic reconstructions offer immediate fixation without the need for healing or biologic ingrowth. They also offer immediate joint stability and early weight bearing. Conversely, these prostheses can be complicated by infection, loosening, hardware failure, and bearing surface wear.6,241 For very young patients, expandable prostheses have recently become available, allowing for limited compensatory growth.389

Allograft-prosthetic composites combine the 2 previously described procedures, by fixing a prosthesis within allograft bone and affixing the composite to the host bone.6,241 This combination enjoys the allograft’s biologic ingrowth potential and its bone restoration, while benefiting from a joint replacement’s stability, modularity, reliability, and longevity. These are often employed in the proximal humerus and the proximal tibia where joint function can be improved by reconstructing tendons directly to intact tendinous insertions on the allograft bone.6,241

The Van Ness rotationplasty is a compromise between an amputation and a limb-salvage procedure, sometimes employed for large tumors of the distal femur.390 It serves to functionally convert an above-knee amputation into a below-knee amputation and has been characterized as an intercalary amputation with sparing of the sciatic nerve and at times, the femoral vessels.390 Once the tumor and surrounding tissues are removed, the distal part of the lower leg is rotated 180 degrees and reattached to the proximal part of the femur, thereby converting the ankle joint into a new knee joint.6,241 A custom prosthesis is eventually fitted to the rotated leg which serves as a below-knee prosthesis. This reconstructive option offers excellent functional outcomes by preserving proprioception and sensation about the foot and decreasing a patient’s energy expenditure.6,241,391-393 It is especially appropriate for skeletally immature patients.391-393 Other surgical options include arthrodeses and the use of vascularized autografts either alone or in combination with allografts; however, these techniques have more limited applications and are far less frequently employed.6,241

Successful surgical treatment of osteosarcoma should be measured by removal of the primary tumor in its entirety and appropriate reconstruction or functional restoration.6,241 At times, this may involve an amputation, which should not be viewed as a failure by any means.394 A well-performed amputation can sustain much higher activity levels and enjoys far fewer complications and revision surgeries when compared with limb-sparing techniques.6,241,395 The psychological impact of an amputation or a Van Ness rotationplasty can often be mitigated after patients meet and speak with others who have already undergone treatment.391-393

For patients with metastatic disease, especially pulmonary metastases, surgery to remove all sites of disease must be considered.295,396 Studies have shown that patients who were rendered free-of-disease surgically have a survival benefit over those whose pulmonary metastases were not completely excised.295,396 Recent reports indicate that despite the reliance on CT scanning as a method for tumor surveillance in the lungs, up to one third of lesions may be missed on CT scanning.397 Therefore, patients who have pathologically proven osteosarcoma of the lungs should undergo exploration of the contralateral lung to rule out and resect radiographically undetected disease.241,397

Treatment of Metastatic and Recurrent Disease

The standard management of patients with metastatic disease follows the same general principles as those who present with localized disease.3-6,8,9,240,241,295 Patients undergo surgery wherever possible to remove all evidence of bulk disease.398,399 Several chemotherapy and surgical timing approaches have been reported for the treatment of these patients. As in patients with localized disease, MAP chemotherapy remains a widely employed treatment backbone, but ifosfamide and etoposide are frequently added in a nonrandomized manner to all patients.3-6,8,9,240,241,295,400 The added efficacy of incorporating ifosfamide and etoposide has not been established. Survival has clearly been enhanced by aggressive treatment designed with a curative intent, but the chances of long-term relapse-free survival are generally less than 20% in patients with more than a single isolated pulmonary nodule.3-6,8,9,240,241,295,401 Given the relatively poor prognosis of these patients, several nonrandomized clinical trials adding novel agents to standard chemotherapy have been performed.3-6,8,9,240,241,295 The new agents being tested will be described further subsequently.

Recurrent disease still occurs in approximately 30% to 40% of osteosarcoma patients who present with localized disease despite a complete surgical removal and intensive chemotherapy treatment.3,4,6,241,401,402 The overall outcome for those patients is poor, and apart from surgery there is currently no universally accepted standard of multimodal treatment.396,403-411 Ferrari et al. reported a projected 5-year postrelapse survival rate of 28% in patients with recurrent osteosarcoma of the extremity, whereas Kempf-Bielack et al. described an actuarial 5-year survival rate of 23% in patients with recurrent osteosarcoma of any site.396,412 Ifosfamide alone and in combination with etoposide have demonstrated response rates of 30% to 40% for the combination in patients with recurrent or metastatic disease.413 As in primary disease, complete surgical resection seems to be a prerequisite for cure. Patients with recurrence in unresectable locations have very little probability of survival.414 Moreover, a long interval between first diagnosis and relapse and a small number of involved sites are favorable prognostic indicators.403-405,408-410,413-415 Treatment with chemotherapy, especially with agents not used during front-line treatment, may offer benefit in some cases. Patients who do not achieve second complete surgical remission and received chemotherapy survived significantly longer than those who did not. However, the role of chemotherapy in relapsed patients who achieved second complete surgical remission is equivocal.403-405,408-410,413-415

Follow-Up and Late Effects

After treatment completion, careful follow-up is required to monitor for signs of recurrence and treatment-related complications. Either surgery or chemotherapy may be associated with long-term disability of some degree.4-6,241 Both limb salvage surgery and amputation may have functional and cosmetic and psychological consequences.387,416-420 Revision surgery may be required in case of prosthesis-related infection, loosening, or breakage. Furthermore, skeletally immature children will require sequential lengthening of their prosthesis.241 Doxorubicin can cause acute and late cardiac toxicity which may become manifest clinically as congestive heart failure or malignant arrhythmias. The risk of cardiac toxicity is related to both dose intensity and total cumulative dose. Lifelong follow-up with serial echocardiograms is essential.421,422 Cisplatin is responsible for high-frequency hearing loss, reported as high as in 11% of the patients.423 However, only a few patients require hearing aids.423 Treatment with cisplatin or ifosfamide may result in chronic renal failure.424 Moreover, gonadal damage may be evident in survivors of osteosarcoma (males more so than females).425 In addition, secondary malignancies (such as leukemia, breast, lung, kidney, central nervous system, and colon cancer) have been observed in 2% to 3% of patients at a median of 7.6 years (1 to 25 years) after primary osteosarcoma treatment.426,427

Summary

Therefore, the standard treatment for patients with nonmetastatic osteosarcoma, although variable, would include an open biopsy to establish the diagnosis. This would be followed by neoadjuvant or induction therapy including cisplatin, doxorubicin, and high-dose methotrexate in North American countries; after about 10 weeks of initial therapy, patients proceed to surgical ablation. Because we have been unable to alter the outcome of poor responders by altering postoperative therapy, continuation of postsurgical therapy with the same agents used preoperatively appears warranted other than in the context of a controlled clinical trial. This approach would be expected to yield a survival rate of approximately 70%. Patients who present with metastatic disease at diagnosis or have recurrent disease have a markedly worse outcome, and long-term cure is only obtained with aggressive approaches to achieve complete resection of all disease sites. The survival of these patients is generally less than 20%.

Emerging Chemotherapy

For the past 15 years, the survival of patients with bone metastases has remained roughly the same despite continuing advances in supportive care, surgical techniques, and even the rapid development of novel therapeutics. It appears that the doses of conventional cytotoxic agents have been maximally utilized in osteosarcoma.4,5,241 With this belief, drug development has shifted from agents like ifosfamide to more “targeted” therapies such as small molecule inhibitors of protein kinases and monoclonal antibodies. The drug development has predominantly focused on patients with metastatic and recurrent osteosarcoma because of their poor prognosis.4,5,241 There are numerous active and completed clinical trials investigating the role that a variety of agents may play in osteosarcoma therapy.4,5,241 Many have appeared to fail, whereas some may have been potentially successful. Some agents have thus far not demonstrated benefit in osteosarcoma (including 17-N-allylamino-17-demethoxygeldanamycin [17-AAG],428 octreotide pamoate long-acting release [OncoLar®],429,430 and ET-743.431 Potential successes include muramyl tripeptide–phosphatidyl ethanolamine (discussed previously),359,364 interferon (mentioned previously),241,368 samarium (mentioned previously),384,385 trimetrexate,241 inhaled granulocyte macrophage stimulating factor,432,433 and inhaled liposomal cisplatin,241 but additional studies may need to be conducted to validate these results. Other studies have been conducted incorporating novel agents to define feasibility, such as the studies of the bisphosphonate, pamidronate, and trastuzumab.241 In the context of these studies, it is difficult to define the potential efficacy of the novel agent. Other clinical studies such as the ongoing studies of the new antifolate, pemetrexed, are too early to make statements with regard to potential efficacy. Other new agents such as the IGF-1 receptor antibodies, VEGF antibodies, and rapamycin have generated variable excitement based on preclinical data and clinical data in other sarcomas, but little data exist for their efficacy in osteosarcoma.241 Selected clinical studies will be briefly presented but space precludes presenting all of the studies as well as detailed descriptions.

Understanding the mechanisms of resistance to agents in osteosarcoma may guide the testing of novel compounds with pharmacologic properties that theoretically circumvent the known mechanisms of resistance. The development of trimetrexate and its studies in osteosarcoma are an example. Methotrexate has been a cornerstone of osteosarcoma therapy, but resistance to methotrexate can develop in multiple ways, and the development of new antifolates has been directed in part from a desire to circumvent the known mechanisms of methotrexate resistance.231,232,234,235,241 Even though trimetrexate and other novel antifolates have been shown to have activity in a wide range of solid tumors in vitro, preliminary studies in osteosarcoma have demonstrated their potential role in therapy.434-436 Results from a completed conventional phase 2 trial in patients with recurrent osteosarcoma showed a 13% response rate (including minor responses) in 38 patients. Myelosuppression (grades 3 and 4) was the most frequently observed toxicity.241 Pemetrexed, another new antifolate that has been shown to be effective in lung cancer and a variety of other solid tumors is now in phase 2 studies in children with recurrent tumors, including osteosarcoma.437-439

Another potentially fruitful avenue of research has been the development of novel delivery mechanisms. Innovations in drug formulation may improve the delivery efficiency of current cytotoxic agents and improve the therapeutic index.440-443 For example, liposomally encapsulated doxorubicin has been shown to be an effective mode of delivery of doxorubicin and has been shown to be safe in a limited number of studies. Its activity in relapsed sarcomas suggests liposomal doxorubicin may be able to overcome tumor resistance mechanisms.241,440-443 A phase 1b/2a study assessing the safety and efficacy of an aerosolized liposomal formulation of cisplatin (Sustained release Lipid Inhalation Targeting [SLIT™] cisplatin, Transave, Inc.) in relapsed osteosarcoma recently closed to patient accrual.241 Preliminary data suggest that aerosolized liposomal cisplatin is well tolerated in heavily pretreated patients. Although an objective response was obtained in a single patient, stable disease or better were noted in 2 of 18 patients while another 2 patients with solitary lung nodules achieved durable remissions with staged thoracotomies and 1 year of therapy.241 For patients with osteosarcoma, this may represent an effective method of delivering a high enough local concentration of cisplatin to overwhelm any mechanisms of resistance without unacceptable systemic toxicity. With respect to inhalation therapy, there is some concern that aeration to the tumor nodules may be poor. At least in the case of osteosarcoma, the nodules will typically be removed as part of the overall treatment strategy at relapse. Therefore, the efficacy of inhalational therapy in osteosarcoma may be better measured in the prevention of additional foci of disease in the lungs. The drawback is obviously the lack of efficacy of inhaled agents against any other sites of disease outside of the lungs.241 Inhalational agents offer the advantages of low systemic toxicity, focused delivery of agents to sites of disease, and high local concentration. Inhalational agents under active investigation include interleukin-2 (renal cell carcinomas), interferon (lung cancer), and granulocyte macrophage colony stimulating factor (GM-CSF; a phase 2 trial of aerosolized GM-CSF has been completed for recurrent osteosarcoma by the Children’s Oncology Group).241

Several investigators demonstrated that overexpression of c-erbB-2, also known as HER2/neu, has prognostic significance in osteosarcoma and that it may play a role in maintaining the malignant phenotype in osteosarcoma, although the data are controversial.107,108,111,116,143,144,146,147,149-153 Preclinical studies of trastuzumab, the humanized monoclonal antibody targeting the HER2 protein, suggested its ability not only to antagonize the function of growth-signaling properties of the HER2 system, but to recruit immune effectors against the target, and to augment chemotherapy-induced cytotoxicity.444-446 Promising clinical data of trastuzumab in HER2 overexpressing breast cancer led to a phase 2 study by the Children’s Oncology Group of trastuzumab in patients with metastatic osteosarcoma whose tumors overexpress HER2.444-447 This study successfully established the feasibility of this regimen which was the primary endpoint, but the efficacy data available at present have been somewhat more difficult to interpret.241

Dysregulated insulin-like growth factor signaling is emerging as an important molecular target for cancer therapeutics development.241 Strategies that inhibit IGF-1 are particularly interesting. As mentioned previously, osteosarcoma has been associated with growth. Therefore, it is not surprising that IGF-1, a major regulator of growth of normal tissues, has been found to be mitogenic in in vitro models of osteosarcoma.104,123,124,126,128,162 Additionally, IGF-1R has been found to be abundantly expressed in osteosarcoma cells, suggesting inhibition of the receptor or its pathway may be effective in osteosarcoma. An early attempt at suppressing the IGF-1 axis was a phase 1 study demonstrating that suppression of serum levels of IGF-1 with sustained release sandostatin (OncoLar®, Novartis) did not produce any tumor responses when combined with tamoxifen.429,430 It is likely that the level of suppression of IGF-1 achieved in this trial was insufficient to produce a biologically relevant effect. Furthermore, suppression of serum IGF-1 levels alone can potentially be ineffective because of the redundant autocrine and paracrine secretion of the growth factors and signaling pathways.429,430 Therefore, targeting the receptor may result in improved inhibition of the IGF-1 signal transduction pathway and thus lead to better cytotoxic effects.241 Using osteosarcoma cell lines, it has been demonstrated that several key signal transduction pathways downstream of the IGF-1 receptor (such as the AKT and MAP kinase pathways) are activated in osteosarcoma, further lending credence to the idea that inhibition of IGF-1 receptor and components of its pathway may be a viable strategy in osteosarcoma. Several new IGF-1 receptor antagonists (predominantly monoclonal antibodies but small molecule inhibitors also exist) are being introduced in phase 1 and 2 studies for sarcomas. As an example, a phase 2 trial of R1507, a recombinant monoclonal antibody to IGF-1R, which includes a stratum for patients with osteosarcoma has already been initiated by the Sarcoma Alliance for Research through Collaboration (SARC).241 Multiple competing trials of IGF-1R antibodies are being performed simultaneously with slightly differing eligibility and response criteria using different monoclonal antibodies. As each of the antibodies is different, epitope specificity, antibody subclass and toxicity, relative efficacy may ultimately need to be clarified. Further testing of these compounds as single agents and in combination with others will need to be done in order to fully assess their role in osteosarcoma treatment.241

Preclinical studies suggested that the mTOR pathway may be a potentially effective target in osteosarcoma.154,155,157,163,166 Rapamycin is widely available, and several new rapamycin analogs (generally with less toxicity than rapamycin) have completed early stage clinical trials in sarcomas. A large phase III trial using the rapamycin analog (AP23573) is underway as a multi-institutional consortium study.241 The vascular endothelial growth factor (VEGF) pathway is another growth factor signaling system recognized as a potential target of therapy in pediatric osteosarcoma, as studies have shown that VEGF expression correlates with metastasis and poor outcome.105,109,112,115 A number of VEGF pathway inhibitors are available now and are being tested in the preclinical arena.241 Encouraging results for AZD2171, a specific VEGF-receptor inhibitor, demonstrated growth inhibition in solid tumor xenograft models including osteosarcoma.448

As researchers shift focus away from the tumor to its interaction with the microenvironment, therapies disrupting that interaction may also lead to new ways of treating osteosarcoma.241 Because these local tumor–microenvironment interactions may be important in supporting tumor growth and metastasis, a therapeutic strategy aimed at inhibiting the tumor microenvironment (such as inhibition of PTHrP and its related pathways) may be efficacious in osteosarcoma.449 Other targets of potential interest include the receptor activator for nuclear factor κ-B (RANK)/RANK-Ligand (RANKL)/osteoprotegrin (OPG) system.120 Using in vitro osteosarcoma models, bisphosphonates have been shown to inhibit tumor growth and to disrupt their interactions with the surrounding microenvironment.450,451 A recent phase 2 study performed at Memorial Sloan-Kettering Cancer Center assessing the role of pamidronate in the treatment of localized osteosarcoma has closed to accrual; early indications are that pamidronate can be safely given in the context of osteosarcoma therapy. However, whether the addition of bisphosphonates to classical 3-drug chemotherapy affords survival benefit remains to be proven in a larger subsequent study.241 More recently, clinical studies have been performed using denusomab, a monoclonal antibody directed to RANKL both as a therapy for osteoporosis and bone metastases. Its role is yet to be defined in osteosarcoma.241

The Potential Clinical Relevance of Osteosarcoma Biology Studies

Recent progress in improving the survival of osteosarcoma patients has been limited. It is hoped an increased understanding of biology will translate into clinical benefit. The clinical goals of osteosarcoma research include identifying prognostic factors that may serve as a basis for stratification of therapy and helping to prioritize clinical trials of new therapeutic agents.4,5,12,276,413 It must be acknowledged that thus far no biologically based validated prognostic factors have been identified. The failure to identify prognostic factors can be explained in a variety of potential ways. A factor must be measured in a sufficiently standardized manner and remain prognostic across a prospective multi-institutional study in order to be clinically relevant. Only factors identified as part of large multi-institutional biology studies are likely to meet these criteria.4,5,12,276,413 For biology efforts to be successful, the establishment and maintenance of large clinically annotated banks of tissue will be critical. A successful model for such an effort comes from the Children’s Oncology Group osteosarcoma tumor bank. The biology study was initiated in 1998, and all osteosarcoma patients aged ð 40 years were eligible for participation. Part of its success can potentially be attributed to the fact that specimen collection was centralized and predominantly performed through an impartial National Cancer Institute funded group, the Cooperative Human Tissue Network. Access to tissue was through an application process and was open to all investigators. Through this effort, the largest reported bank of osteosarcoma-related biological materials has been established and a large number of studies using this material are underway.4,5,12,276,413

The Pediatric Preclinical Testing Program

A tremendous clinical need is the rapid identification of new therapies that are effective in the treatment of osteosarcoma. In order to do so, increasing emphasis is being placed on preclinical laboratory studies of chemotherapy effectiveness in various tumor model systems.4,5,12,276,413 The rationale for this approach includes the following: if a drug targets a pathway that is central to a tumor’s viability, ie, if it is so-called “gene addicted,” we would expect it to be efficacious. As long as normal cells were less dependent upon the pathway, a therapeutic index should exist. Although few drugs are developed specifically for sarcomas, pathways central to some sarcomas may overlap with those of more common cancers. Numerous new drugs exist, and there are too few osteosarcoma patients to test them all clinically.4,5,12,276,413 The Pediatric Preclinical Testing Program is one such effort underway to facilitate the introduction of new, active agents into clinical trials for all childhood cancers. With a consortium of laboratories in the United States and abroad, the Pediatric Preclinical Testing Program is able to quickly screen a large number of agents using in vitro and in vivo models.448,452-459 Preclinical testing potentially may predict the activity of new agents in patients with childhood cancers, allowing the rational design of clinical trials utilizing new agents that can presumably lead to the identification of active agents more rapidly. Some believe that the value of the preclinical testing needs to be validated as accurately representing responses in human clinical trials prior to utilizing the information as a means of prioritizing clinical trials. Others believe in the absence of other data that preclinical testing should be used as a basis of prioritization as it is more likely to be predictive than intuitive or random selection of new agents for clinical trials in osteosarcoma.241 Regardless of how the data are utilized, the Pediatric Preclinical Testing Program has generated a large amount of data that have rapidly been published.448,452-459 The group has evaluated several standard and novel agents including cyclophosphamide, vincristine, topotecan, 19D12 (Anti-IGF-1R antibody), AZD2171 (a specific inhibitor of VEGF-receptor), AZD6244 (MEK1/2 inhibitor), bortezomib (proteasome inhibitor), ABT-263 (a Bcl-2 inhibitor), MLN8237 (aurora A kinase inhibitor), rapamycin, vorinostat (histone deacetylase inhibitor), lapitinib (EGFR and ErbB2 inhibitor), and sunitinib. Thus far, of all of the novel agents tested—19D12, AZD2171, and rapamycin—have been among the most effective for the treatment of osteosarcoma.448,452-459

Other appealing model systems exist, most notably pet dogs that develop osteosarcoma spontaneously.460,461 The osteosarcomas in canines are similar to those that occur in humans and have the advantage of arising spontaneously and therefore of being heterogeneous. The high incidence of osteosarcoma in dogs and the more accelerated time course for progression of their disease facilitates the conduct of clinical trials in dogs analogous to those in humans. Linkages have increasingly been developed between the veterinary oncology consortiums and the oncology cooperative groups.460,461 Most notably, comparative oncology programs exist in the context of the National Cancer Institute and the Children’s Oncology Group, with osteosarcoma a major interest.460,461

Though rare, osteosarcoma is a very treatable and curable disease. With combination chemotherapy and aggressive surgical resection, the majority of patients can expect to be cured of their disease. Chemotherapy and surgery remain the cornerstones to this success. However, as survival for patients with this disease has not improved in 15 years, there is much need for improvement and innovation. The current international treatment protocol for osteosarcoma (EURAMOS-1) demonstrates the cooperation (national and international) that is necessary to quickly address the unanswered clinical questions in this disease. Moving forward, the large number of novel agents entering clinical trials and an enhanced understanding of osteosarcoma’s biology may have a major impact on the way osteosarcoma will be treated in the future.

Ewing sarcoma (ES) is the second most common bone tumor in children and adolescents. It was named for James Ewing, a pathologist who first described the tumor in 1921, emphasizing its distinction from osteosarcoma based upon enhanced radiosensitivity and a propensity to involve flat bones of the axial skeleton.1 In the early 1980s, both Ewing sarcoma and peripheral primitive neuroectodermal tumor (PNET) were found to contain identical t(11;22)(q24;q12) translocations.2,3 Based upon the shared translocation, and similar clinical behaviors, Ewing Sarcoma Family of Tumors (ESFT) is now considered one disease entity that includes classical ES, atypical ES, peripheral primitive neuroectodermal tumor, neuroepithelioma, and Askin tumor (an ESFT of the chest wall). It should be emphasized that peripheral PNETs are entirely different from PNETs arising in the central nervous system which do not bear the t(11;22) translocation and are not considered part of the ESFT category.

Epidemiology, Pathophysiology, and Genetics

ESFTs occur in approximately 2 to 3 per million/year in persons less than 20 years4 with a peak in the second decade of life, although they have been reported in infants and occur with a substantial incidence in adults, especially prior to age 40. For unknown reasons, ESFT are extremely rare among individuals of African and Asian heritage. This is in contrast to osteogenic sarcoma, which has a relatively equal race distribution.4 Despite this dramatic ethnic predilection, neither specific genetic nor environmental risk factors for the development of ESFT have been identified. For instance, unlike osteosarcoma, which occurs more commonly following irradiation, there is no evidence for an increased incidence of ESFT following irradiation, and there is no evidence that ESFT occurs more frequently in patients with defined cancer predisposition syndromes. Importantly, however, family members of ESFT patients have an increased incidence of neuroectodermal, stomach, and breast malignancies, and survivors of ESFT show a high rate of second malignant neoplasms, suggesting that as yet undefined genetic factors may predispose to ESFT.5

The cell of origin that gives rise to ESFT is also unknown, with primitive neural cells implicated historically, and recent studies suggesting that mesenchymal stem cells may give rise to the disease.6,7 The classic translocation t(11;22)(q24;q12), or another related translocation, occurs in greater than 95% of ESFT.8 ESFT-associated translocations join the Ewing sarcoma (EWS) gene located on chromosome 22 to an ets-family gene, most commonly FLI1 (Friend Leukemia Insertion), located on chromosome 11.9 The 68-kDa protein produced by the EWS-FLI1 fusion transcript functions as an aberrant transcription factor and plays a critical role in initiating and sustaining ESFT. Expression of this molecule can transform mouse fibroblasts,10 and reduction of EWS-FLI1 expression induces death of ESFT cell lines.11-13 Such studies have established the EWS-ets translocation as a compelling therapeutic target. However, clinically applicable therapies to successfully prevent expression of EWS-ets or interfere with downstream oncogenic events induced by the fusion transcript have proven elusive thus far.14

Clinical Features and Differential Diagnosis

ESFTs most often arise in bone and have been reported in virtually every bone, but can also occur in soft tissues without bony involvement. Unlike osteosarcoma, which usually arises in the metaphysis of long bones, ESFT arises more frequently in flat bones (eg, pelvis, vertebrae, ribs, skull), and when it arises in long bones, it commonly involves the midshaft or diaphysis. ESFT patients usually present with pain or a palpable mass, and ESFT should be included in the differential diagnosis of any bone or soft tissue mass in patients from infancy through early adulthood. Unfortunately, many patients ultimately diagnosed with ESFT often describe a history of chronic pain for many months before the correct diagnosis is made. This is especially true for pelvic ESFTs when a palpable mass is not present and the presenting symptoms of neuropathic, radicular pain or vague back discomfort are often attributed to sports-related or other trivial injuries in adolescents and young adults. It is important, therefore, that children and young adults who experience persistent or recurring pelvic, back, or radicular pain undergo appropriate radiographic evaluation to rule out a malignancy involving the spine, pelvis, or extremity. Radionuclide bone scan may be particularly helpful in identifying a neoplastic cause of chronic pelvic, back, or radicular pain. Occasionally, ESFTs present with vertebral involvement, and in such cases, an associated extradural mass can compress the spinal cord and induce paraparesis or paraplegia, with sphincter dysfunction. Tumors adjacent to the spinal canal require immediate magnetic resonance imaging (MRI) scanning because intervention is sometimes needed to prevent neurologic deterioration, which can occur rapidly if spinal cord compression is present.

Classic radiologic findings of ESFT include permeative bone destruction with a moth-eaten appearance, elevation of periosteum with periosteal reaction (Codman triangle) or a lamellar (onion skin) lesion on plain radiographs, which results from cortical destruction followed by reparative cortical bone laid down by reactive osteoblasts (Fig. 454-1). Computerized tomography (CT) scans provide optimal imaging to assess the degree of cortical bone involvement, whereas MRI accurately establishes the extent of soft tissue and marrow involvement (Fig. 454-1). The differential diagnosis of destructive bony lesions should include osteogenic sarcoma, other sarcomas arising in bone (malignant fibrous histiocytoma, chondrosarcoma, fibrosarcoma), giant cell tumors, benign neoplasms, eosinophilic granuloma, lymphoma, or metastases from another primary neoplasm. For patients with a soft tissue mass without bony involvement, the differential diagnosis includes rhabdomyosarcoma, neuroblastoma, other soft-tissue sarcomas, lymphoma, or benign neoplasms. Systemic symptoms of fever and weight loss can occur as part of the presenting symptom complex in patients presenting with ESFT, especially with large primary tumors or metastatic disease.

Diagnostic Evaluation

Children believed to have a malignant bone or soft-tissue tumor should be referred to a pediatric care center with experience and expertise in the treatment of sarcoma. When ESFT is suspected clinically, consultation with a pediatric oncologist and an orthopedic oncologist should occur prior to biopsy. Selection of the biopsy site and approach should be made in consultation with the orthopedic oncologist, because an inappropriate biopsy can adversely impact future options for resection or radiation therapy. The diagnosis can often be made on routine and immunohistochemical stains; however, there is increasing emphasis on molecular identification of the t(11;22) or a related translocation to confirm the diagnosis. Molecular studies often require specific handling procedures; therefore, the pathologist should be alerted prior to the biopsy to ensure appropriate handling and triaging of the tissue.

Histologically, ESFT is a small round blue cell tumor that can span a spectrum from undifferentiated Ewing sarcoma characterized by sheets of small round blue cells to the more differentiated peripheral primitive neuroectodermal tumor wherein pseudorosettes are seen. Immunohistochemical studies show diffuse membranous staining for CD99 (MIC2)15 in greater than 90% of ESFTs. Although robust CD99 expression can reliably rule out neuroblastoma, nonmembranous CD99 staining is not infrequent in other small round blue cell tumors such as rhabdomyosarcoma and lymphoma; therefore, CD99 expression is not specific or diagnostic as a single finding for ESFT. Muscle and lymphoid markers should routinely be performed to rule out rhabdomyosarcoma and lymphoma, respectively, as well as other immunohistochemical studies deemed necessary by the consulting pathologist. Molecular identification of a ESFT-specific translocation in a tumor with the appropriate histology is pathognomonic for the diagnosis, and this can be accomplished using standard cytogenetics, FISH or RT-PCR (reverse transcription polymerase chain reaction).

Once a histologic diagnosis of ESFT is made, it is important to determine the anatomic extent of the primary tumor using CT and MRI scanning, so that effective delivery of therapies for local control can be rendered. Although such therapies are typically delayed for several weeks following initiation of multiagent chemotherapy, adequate local control must be based upon the initial extent of the primary tumor, rather than on the postchemotherapy tumor extent. The diagnostic evaluation at the time of diagnosis should also distinguish patients with clinically localized ESFT from patients with macrometastatic disease, because this has important effects on prognosis and potentially on the systemic therapy administered. Therefore, all patients with ESFT should also undergo CT scanning of the chest to rule out pulmonary metastases (Fig. 454-1), radionuclide bone scan or positron emission tomography (PET) scan to rule out bony metastases, and bilateral bone marrow aspiration and biopsy to rule out marrow metastases.

Treatment

Historical studies demonstrated that aggressive local therapy alone, such as amputation of distal lesions, cured less than 10% of patients with Ewing sarcoma due to metastatic relapse. Because of this, all patients diagnosed with ESFT should be assumed to have disseminated micrometastases, even in the absence of radiographic findings. Cure requires both effective local and systemic therapy. Therefore, all patients with ESFT should receive multimodal therapy, which includes dose-intensive, multiagent chemotherapy with radiation or surgery for local control.

Therapy for ESFT has evolved based upon sequential results from a series of cooperative group studies conducted primarily in North America and Europe. Currently, approximately 70% of patients who present without clinical or radiographic evidence for metastatic disease experience 5-year disease-free survival. Vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide are the most active single agents in ESFT and form the backbone of nearly all initial chemotherapy regimens for this disease.16 In general, dose-intensive regimens have shown improved survival compared to less-intensive regimens, with the most recent Children’s Oncology Group study demonstrating improved survival for patients with nonmetastatic ESFT when chemotherapy is administered on an every-2-weeks schedule.17

In addition to dose-intensive, multiagent, cytotoxic therapy, cure of ESFT requires effective treatment of the primary tumor. This can be accomplished with radiation therapy, surgery, or a combination of both. ESFT is highly radioresponsive, and radiotherapy is considered a standard option for definitive local control. Radiotherapy can be used as an alternative to disfiguring surgery such as amputation. Alternatively, surgery can provide good local control rates if adequate surgical margins are obtained. In general, patients who undergo surgery as therapy for the primary tumor have improved survival. However, because patients with smaller or distal tumors typically undergo surgery, whereas patients with larger or axial tumors typically undergo primarily radiation therapy, the differences observed relate, at least in part, to selection bias. Importantly, however, because radiation therapy for ESFT is associated with a risk for second malignancies within the radiation field, surgery is generally preferred for local control when adequate tumor margins can be accomplished and significant morbidity or functional deficits will not result. Postoperative radiotherapy should be administered when surgical margins are close or positive.

Complications

Toxicity is an important consideration in the therapy of ESFT because the regimens used are dose intensive. For this reason, multidisciplinary (infectious disease, surgical, radiation therapy, nutritional, psychological) care is essential to optimize patient management. Severe neutropenia occurs in most patients, despite G-CSF (granulocyte colony stimulating factor) support, and serious complications related to fever, neutropenia, and mucositis commonly occur. In addition, cumulative irreversible cardiotoxicity from doxorubicin is a potential risk for all patients and requires a careful approach to minimize the extent of this late effect. To that end, essentially all ESFT protocols limit the total doxorubicin dose for treatment of newly diagnosed patients at 375 to 450 mg/m2; all patients are followed closely for cardiac function throughout therapy. Doxorubicin is omitted if diminished cardiac function is observed, and most centers use either prolonged infusions of doxorubicin or concomitant therapy with the cardioprotectant dexrazoxane (ICRF-187) to maximize doxorubicin dose intensity and minimize the risk for long-term morbidity related to anthracycline exposure.18 Ifosfamide commonly induces renal tubular damage with electrolyte wasting which in the extreme can result in Fanconi syndrome and hypophosphatemic rickets. Vincristine induces a potentially irreversible peripheral neuropathy which may be accompanied by difficulty in dorsiflexion or limited fine motor coordination, and this agent is typically held when functional significant peripheral nerve damage is present. Alkylating agents used in the treatment of ESFT result in a substantial rate of infertility and a risk for treatment-induced acute myeloid leukemia and myelodysplastic syndrome, which ranges from 1% to 3%. Finally, the incidence of second malignancies occurring in the radiation field of patients with ESFT is approximately 5%, with a relationship observed between higher radiation doses and an increased rate of second malignant neoplasm.

Prognosis

No widely accepted staging system exists for ESFT. However, the most important prognostic factor is the presence or absence of clinically detectable, metastatic lesions. For patients with localized disease, survival approaches 70%. Among patients without metastasis, those with larger primary tumors and tumors involving the axial skeleton fare worse than those with smaller more distal tumors. However, aggressive multiagent chemotherapy regimens have diminished the importance of tumor site and size on outcome. Poor histologic response to chemotherapy and the presence of specific molecular abnormalities involving the p53 pathway (eg, p53 mutations or p16INK4a abnormalities) confer a poorer prognosis.19 Older age is also a negative prognostic variable for patients with localized disease, although the reason for this remains unclear.

ESFT patients with clinically evident metastases at initial diagnosis continue to have a poor prognosis despite the administration of dose intensive, multiagent chemotherapy which has improved outcome for patients with localized disease.20 Patients with metastases confined to the lungs may represent a group of patients with better prognosis than patients with bone or bone marrow metastases. For example, in the European Intergroup Ewing Sarcoma Study (EICESS 92), patients with pulmonary-only metastases had a 40% 4-year disease-free survival when treated with whole-lung irradiation in addition to multiagent chemotherapy.21 In contrast, patients with bone or bone marrow metastases have 5-year survival rates of < 20% in most series.

Future Directions

To improve survival and reduce morbidity, novel combinations of established chemotherapeutic agents and molecular targeted therapies are being tested in ESFT. Because EWS-FLI1 is a specific molecular target, future therapies are likely to focus on inactivating EWS-FLI1 function, although no drugs are currently available.14 The insulin-like growth factor type I receptor is critical for transformation and growth of ESFT.22 New inhibitors of IGF-IR signaling have shown promise in early phase trials, and studies are currently underway to establish true response rates. Future studies will seek to incorporate these therapies along with standard, cytotoxic therapy. The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) can activate tumor necrosis factor–modulated apoptotic pathways in ESF.23,24 TRAIL-activated apoptosis occurs through caspase-8 in ESFT, and most ESFT cell lines express caspase-8. TRAIL may represent a biologic approach to inducing apoptotic cell death in ESFT.

Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, representing approximately 5% of all cancers among children and adolescents.1-5 Approximately 350 new cases are diagnosed each year in the United States, for an annual incidence of 4.3 per million children and adolescents younger than 20 years.1 Although RMS can occur in adolescents and adults, two thirds of patients are less than 10 years of age.1,4 The incidence of RMS is slightly higher in males.1 With current multimodal therapy (including chemotherapy, radiotherapy, and surgery), approximately 70% of children with RMS can be cured.2-6

Genetics and Epidemiology

A small fraction of RMS cases occur as part of recognizable genetic syndromes, either as an inherited gene in an affected family or as a new germline mutation. These syndromes can be divided into those with only tumor susceptibility and those with both tumor susceptibility and nonneoplastic effects. The former category includes both Li Fraumeni and hereditary retinoblastoma syndromes, which are caused by mutations in the TP53 and RB1 tumor suppressor genes, respectively.7 In Li Fraumeni syndrome, which is characterized by susceptibility to a heterogeneous group of cancers, RMS occurs relatively frequently in addition to other soft tissue sarcomas, breast cancer, brain tumors, and acute leukemia.8 In contrast, RMS occurs less frequently as a secondary tumor in hereditary retinoblastoma syndrome, typically presenting in or near the radiation field following treatment of the primary retinoblastomas.9

Costello syndrome and neurofibromatosis type-1 are syndromes with tumor susceptibility and nonneoplastic effects that are caused by mutations of genes in the RAS signaling pathway—HRAS and NF1, respectively.10 In addition to developmental effects involving the skin and brain, these two syndromes are characterized by a high incidence of benign tumors (skin papillomata and neurofibromas, respectively) and a lower incidence of malignant tumors. RMS is the most common cancer in Costello syndrome, whereas RMS occurs less commonly than malignant peripheral nerve sheath tumors in neurofibromatosis type-1.

In a final group of other tumor susceptibility syndromes with nonneoplastic effects, there are only a few reported RMS cases. These conditions include Beckwith-Wiedemann, Gorlin, and Rubinstein-Taybi syndromes, which are linked to dysregulated imprinted genes in the 11p15 chromosomal region, PTCH mutations, and CREBBP mutations, respectively.7,11,12 In the first two conditions, RMS occurs infrequently relative to a frequent cancer type (Wilms tumor and basal cell carcinoma, respectively), but in the latter, RMS and all other tumors occur infrequently.10,13,14

Pathology and Genetics

The tumors of the RMS family have in common a poorly understood relationship to the skeletal muscle lineage and often can be diagnosed based on evidence of this differentiated phenotype. RMS is then further subclassified into two main histopathologic subtypes—alveolar (ARMS) and embryonal (ERMS).15 ARMS is characterized by small cells with round nuclei and scant cytoplasm. Aggregates of ARMS tumor cells are typically divided by fibrovascular septae, and spaces often form within these aggregates to produce an “alveolar” appearance. In contrast, ERMS tumor cells can show varying cellular features that parallel the process of myogenic differentiation, ranging from small round cells to large elongated cells with eccentric oval nuclei and abundant eosinophilic cytoplasm. Furthermore, the overall density of ERMS tumor cells often varies in these tumors with highly cellular areas intermixed with low cellular areas in a loose myxoid stroma.

At the cytogenetic level, most ARMS cases can be distinguished from ERMS as well as other pediatric solid tumors by the presence of recurrent chromosomal translocations.16 The most frequent translocation involves chromosomes 2 and 13 [t(2;13)(q35;q14)], and a less frequent variant involves chromosomes 1 and 13 [t(1;13)(p36;q14)]. These translocations juxtapose portions of the PAX3 or PAX7 gene on chromosome 2 or 1, respectively, with a portion of the FOXO1 or (FKHR) gene on chromosome 13 to generate a PAX3-FOXO1 or PAX7-FOXO1 fusion gene. Each fusion gene is transcribed into a fusion transcript that is in turn translated into a fusion protein. These fusion proteins function as aberrant transcription factors to alter the transcriptional program of the target cell and thereby contribute to the oncogenic phenotype.

In contrast to the translocations found in ARMS, there are no recurrent structural chromosomal changes in ERMS. However, loss of one of the two alleles of many chromosome 11 loci t is a much more frequent occurrence in ERMS than in ARMS.17 This allelic loss localizes to chromosomal region 11p15.5, which is the same region to which the cancer susceptibility gene associated with Beckwith-Wiedemann syndrome and the allelic loss events in other pediatric solid tumors such as Wilms tumor have been localized. In this region, there are multiple genes whose expression is epigenetically regulated in a parent-of-origin-specific fashion by genomic imprinting. A scenario is proposed in which one allele of a tumor suppressor gene in this region is physiologically inactivated by imprinting, and the second allele is removed by the allelic loss event, thereby inactivating both tumor suppressor alleles and promoting an oncogenic effect.

Clinical Features and Differential Diagnosis

The most common sites of presentation for RMS are the head and neck (including the orbit and parameningeal sites), genitourinary system (including bladder/prostate and paratesticular sites), extremities, and trunk.2-6 The presenting signs and symptoms of RMS are diverse because RMS can originate at any anatomic site (Table 455-1). For example, tumors of the neck or extremity are usually detected as an enlarging mass. Orbital masses can cause proptosis and diplopia. Parameningeal tumors may cause cranial nerve dysfunction (including ophthalmoplegia and facial nerve palsy), external ear drainage, and nasopharyngeal obstruction. Paratesticular tumors present as a scrotal mass. Pelvic or bladder/prostate tumor can present with urinary obstruction and hematuria. The differential diagnosis of a soft tissue mass also varies by anatomic site and presenting symptoms. Perineal and neck masses can result from infectious causes, such as bacterial abscess. Nasopharyngeal masses can appear similar to sinusitis. Benign soft tissue masses, such as hematoma, hemangioma, or fibromatosis, can also mimic RMS.

Diagnostic Evaluation

The investigation of a soft tissue mass in a child or adolescent includes a careful physical examination and diagnostic imaging to define the size, location, and local invasiveness of the primary mass and to detect regional lymphatic metastases. Computerized tomography (CT) and magnetic resonance imaging (MRI) are the most common imaging modalities to assess the local and regional extent of disease. Because of the risk of distant metastatic spread, chest CT, bone scan, and bone marrow aspiration and biopsy are routine staging evaluations for all patients with RMS. Definitive diagnosis is established with a biopsy or resection of the primary mass or biopsy of a metastatic site, depending upon the site’s accessibility and risk of a surgical procedure.

The determination of the presence of RMS within a tissue sample involves several possible diagnostic technologies. Evaluation commences with histopathological evaluation of the sample and detection of a neoplastic population of cells with cytologic and architectural features resembling the ARMS or ERMS patterns described above.15 In some cases, evidence of myogenic differentiation can be seen microscopically by the presence of elongated eosiniphilic “strap-like” cells that may have cross striations or multinucleation. More subtle evidence of myogenic differentiation is provided by immunohistochemical stains directed against muscle-specific proteins such as desmin, or the myogenic transcription factors MyoD and myogenin. Additional evidence of muscle differentiation can also be detected by electron microscopic evaluation of the presence of myofilaments.

To help identify the majority of ARMS cases, tissue samples can also be evaluated for the presence of the 2;13 or 1;13 chromosomal translocations. Though tumor cells can be analyzed by classical cytogenetics, molecular-based testing is faster, more accurate, and less expensive. One popular molecular technology for identifying these translocations is to assay for the PAX3-FOXO1 and PAX7-FOXO1 fusion transcripts in RNA isolated from tissue by the reverse transcription-polymerase chain reaction.18 An alternative technology is to assay for the PAX3-FOXO1 and PAX7-FOXO1 fusion genes in tumor cells or nuclei by fluorescence in situ hybridization. Comparative studies have shown that the results of these two molecular technologies are similar,19 and that ~60% of ARMS cases are PAX3-FKHR-positive, ~20% are PAX7-FKHR-positive, and ~20% are fusion-negative.18

Treatment

Treatment for RMS is risk adapted, with less radiotherapy and chemotherapy for patients at lower risk for disease recurrence and more therapy for patients at higher risk for recurrence.4-6,20 Factors that most influence success of therapy include presurgical stage (determined by anatomic site, primary tumor size, and regional nodal spread), postsurgical group (defined by the extent of resection), presence of distant metastases, and histologic subtype. These factors establish three risk groups: low-risk, intermediate-risk, and high-risk RMS (Table 455-2).

RMS is uniformly treated with systemic chemotherapy. For patients with localized RMS who have initial complete surgical resection, the role of chemotherapy is to treat micrometastatic disease. For initially unresected tumors, chemotherapy can improve the success of local treatment (either radiotherapy or a delayed surgery) to prevent a recurrence at the primary site. Routine use of systemic, multiagent chemotherapy has resulted in impressive improvement in survival rates over the last 30 years. In North America, the most frequently used chemotherapeutic agents are vincristine, dactinomycin, and cyclophosphamide (VAC). Additional agents, including doxorubicin, ifosfamide, and etoposide, are sometimes used to treat patients with metastatic RMS with variable success.4-6,21,22

In addition to chemotherapy, the management of the primary tumor mass includes local radiation therapy and surgical excision.2-6 The selection of either modality depends on the primary site, local invasiveness, and anticipated long-term morbidity of treatment. When feasible without risk of loss of function, initial complete surgical resection is preferred. Completely resected ERMS can be treated with less-intensive chemotherapy, potentially with the omission of radiotherapy. Completely resected ARMS can be treated with lower-dose radiotherapy. Paratesticular primaries and some small, superficial head and extremity primaries are usually treated with surgical excision at diagnosis. Other sites, such as bladder/prostate or vaginal primaries, usually are resectable but with significant long-term morbidity. Parameningeal sites are almost never amenable to resection either at diagnosis or after initial chemotherapy and are usually treated with radiotherapy as the sole modality of local control. Because most children with RMS have immature skeletons, the risk of altered growth after radiotherapy is a consideration in selecting the modality of local treatment. Comparing results from North America (where radiotherapy is used as the primary mode of local control) to those in Europe (where radiotherapy is used more selectively), radiotherapy appears to be more effective in preventing recurrence and improving survival.22,23

Short- and Long-Term Complications

The treatment of RMS is associated with significant short- and long-term toxicity. Multiagent chemotherapy, such as VAC, can cause reversible nausea, alopecia, mucositis, peripheral neuropathy, and bone marrow suppression, with significant risk of life-threatening infection.3 Routine supportive measures can reduce the risk of serious acute side effects. For example, administration of hydration and MESNA can prevent hemorrhagic cystitis that occurs with cyclophosphamide. Prompt use of empiric antibiotics for febrile neutropenia reduces the risk of fatal bacterial infections. Long-term complications of chemotherapy include infertility (due to high cumulative cyclophosphamide doses) and secondary leukemia. Because most children with RMS have not completed linear growth, conventional radiation therapy can impair growth of muscle and bone within the radiotherapy treatment volume. Radiation to the parameningeal site can lead to long-term neurocognitive impairment and growth hormone deficiency and hypothyroidism due to radiotherapy to critical brain structures. Surgery can also cause long-term sequelae, including amputation for an extremity or cystectomy for bladder/prostate RMS.

Prognosis

For patients without distant metastases, more than 70% of patients will become long-term survivors, with the predicted outcome dependent upon the risk group. The outcome for low-risk patients is particularly favorable, leading to further attempts to limit the extent of treatment, potentially reducing the risk of long-term sequelae. The intermediate-risk patients are treated with moderately intensive chemotherapy and are candidates for clinical trial enrollment to test the addition of new chemotherapeutic agents to improve outcome. In contrast, only 30% of patients with metastatic disease survive long term. High-risk patients are candidates for more innovative therapies to improve their prognosis, including the incorporation of investigational chemotherapeutics. Current research in the treatment of RMS includes the identification of novel agents that target biologic pathways critical to the growth of RMS and functional imaging to determine response to treatment and allow subsequent tailoring of therapy.