77_ftp

PERSPECTIVE JJBMR
Osteosarcoma
Richard Gorlick1 and Chand Khanna2

1Department of Pediatrics and Molecular Pharmacology, The Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA2Tumor and Metastasis Biology Section, Pediatric Oncology Branch, Center for Clinical Research, The National Cancer Institute,Washington, DC, USA

ABSTRACTIt has been difficult to identify the molecular features central to the pathogenesis of osteosarcoma owing to a lack of understanding of

the cell or origin, the absence of identifiable precursor lesions, and its marked genetic complexity at the time of presentation.

Interestingly, several human genetic disorders and familial cancer syndromes, such as Li-Fraumeni syndrome, are linked to an increased

risk of osteosarcoma. Association of these same genetic alterations and osteosarcoma risk have been confirmed in murine models.

Osteosarcoma is associated with a variety of genetic abnormalities that are among the most commonly observed in human cancer; it

remains unclear, however, what events initiate and are necessary to form osteosarcoma. The availability of new resources for studying

osteosarcoma and newer research methodologies offer an opportunity and promise to answer these currently unanswered questions.

Even in the absence of a more fundamental understanding of osteosarcoma, association studies and preclinical drug testing may yield

clinically relevant information. � 2010 American Society for Bone and Mineral Research.

Introduction

Osteosarcoma is a well-defined clinical entity with a

characteristic radiographic appearance, histologic features

that can be readily recognized by pathologists, a relatively

consistent spectrum of clinical presentations, and established

standard treatments. These features have been the subject of

many prior book chapters and reviews and are very briefly

summarized in Table 1.(1–6) A number of recent reviews have

focused on the genetic complexity of osteosarcoma, lamenting

the inability to use the numerous abnormalities present in

tumors either for diagnostic purposes or for prognostication.(5,6)

They have highlighted the inability to identify a precursor lesion

and perhaps even the cell of origin, which has prevented the use

of many laboratory methods employed to study malignancies

with a clear multistep progression such as colon cancer. Despite

these limitations, progress has been made through the use of

epidemiologic features, genomic and molecular characteriza-

tions of available tumor samples, consideration of human

predisposition syndromes, and the study of osteosarcoma in

animal models. A consensus of these studies suggests that p53

and Rb gene/pathway dysregulation is central to the formation

of osteosarcoma in human patients and animal models.(5–7) It is

unclear, however, how p53 and Rb pathway alterations are linked

to the initial development or progression of osteosarcoma.

Received in original form November 16, 2009; revised form January 31, 2010; acce

Address correspondence to: Richard Gorlick, MD, Associate Professor of Pediatrics

University, Department of Pediatrics, Children’s Hospital at Montefiore, 3415 Bainb

E-mail: [email protected]

Journal of Bone and Mineral Research, Vol. 25, No. 4, April 2010, pp 683–691

DOI: 10.1002/jbmr.77

� 2010 American Society for Bone and Mineral Research

Etiology of Osteosarcoma

An extensive list of genetic abnormalities and environmental

exposures, with examples, including fos overexpression and

radiation exposure, has been associated with the development

of osteosarcoma in laboratory models as well as humans.(1–6)

Interestingly, a single recurrent genetic event does not seem to

define this cancer. A unifying approach to bring these

multiple genetic risk factors together with epidemiologic and

association data includes grouping gene or gene families into

two categories: those which drive proliferation (growth) and

those associated with an inability to repair DNA damage with

associated loss of cell cycle regulatory control. This is

summarized in a simplified manner in Table 2. Since both

growth and loss of regulatory control are intrinsic properties of

all cancers, it is not surprising that most osteosarcoma features

can be categorized in this manner. Indeed, when one reviews the

list of associated genetic abnormalities, it is clear that the vast

majority of deranged oncogenes and tumor-suppressor genes

associated with osteosarcoma are also common in the most

prevalent cancers. A corollary assessment of this same result is

that a distinctive and osteosarcoma-specific genetic dysregula-

tion has not been found and, as such, suggests that the

underlying and primary cause of osteosarcoma is a defect in one

of these basic processes, that is, cell proliferation and cell repair.

pted February 25, 2010. Published online March 4, 2010.

and Molecular Pharmacology, Albert Einstein College of Medicine of Yeshiva

ridge Avenue, Rosenthal, 3rd Floor, Bronx, NY 10467, USA.

683

Table 1. Clinical Features of Osteosarcoma

Histologic

appearance

Malignant spindle cell tumor that

produces osteoid

Radiographic

appearance

Lytic and blastic bone lesion classically

described as a ‘‘sunburst’’; periosteal

elevation related to a soft tissue mass

producing a ‘‘Codman’s triangle’’

Clinical

presentation

Pain most common symptom

Location Any bone in the body but most

commonly metaphyseal portion of

appendicular bones; approximately

50% arise around the knee, with the

proximal humerus the next most

common site

Age Bimodal age distribution, with first and

larger peak incidence in the second

decade of life

Dissemination Metastases to lungs and other bones;

approximately 80% present with

localized disease, and in

approximately 90% of patients with

metastatic disease, it will be in the

lungs only

Treatment Surgery to resect all sites of bulk disease

for cure; all high-grade osteosarcomas,

regardless of stage, are treated with

chemotherapy, which most typically

includes cisplatin, doxorubicin, and

high-dose methotrexate; the vast

majority of osteosarcomas in younger

individuals are high grade

As an example, abnormal b-catenin signaling resulting from the

loss of the adenomatous polyposis coli (APC) gene is believed to

drive the formation of colon adenomas, with the end result being

over 200,000 cases per year of colon cancer in the United

States.(8,9) Osteosarcoma, in contrast, occurs in less than 1000

patients per year.(10,11) Therefore, these oncogenic events do not

uniquely define osteosarcoma, nor is the frequency at which

these derangements occur likely to define the incidence of this

disease.

What sets osteosarcoma apart from other malignancies in

large part are the features that are immediately apparent: the

organ of origin and the age of peak onset.(10,11) Although

osteosarcoma comprises less than 1% of cancers diagnosed in

the United States, it is the most common primary malignancy of

bone.(10,11) Bone is an unusual site of cancer formation, andmany

of the unique properties of this disease may be related to either

the cell of origin or unique features related to that environment.

Osteosarcoma has its highest incidence during the second

decade of life.(10,11) This is a period of time in which the incidence

of cancer is low, and although surpassed in incidence by

leukemia and lymphoma, it is one of the more common cancers

during adolescence.(1,3,4) The vast majority of osteosarcomas in

684 Journal of Bone and Mineral Research

children, adolescents, and young adults are high grade and

begin in the intramedullary space of metaphyseal locations in

long bones of the lower extremity, suggesting a relationship to

expanding growth plates.(1,3,4) After a lower incidence in

individuals 25 to 59 years of age, the incidence of osteosarcoma

rises once again in individuals over age 60 to approximate the

incidence in adolescence.(10,11) When osteosarcoma in older

individuals is compared with that in adolescents, a greater

proportion is found to have tumors in axial sites, low-grade

tumors, surface lesions, and rare presentations such as at

extraosseous sites.(10,11) These differences in presentation may

suggest that the underlying pathogenesis of these entities is not

identical independent of epidemiologic features such as Paget’s

disease and prior radiation exposure, which are risk factors

unique to older patients.(10,11)

Osteosarcoma arises from a mesenchymal cell that has or can

acquire the capacity to produce osteoid.(1,3,4) Cells of this type

exist in multiple compartments and include the adherent cell

populations obtained in a bone marrow aspiration, hence the

intramedullary space within bones, with cells of this type

generally referred to as mesenchymal stem cells.(12) They also

include osteoblasts, which are concentrated along both the

endosteal and periosteal surfaces of the bone and involved in

fracture repair and remodeling. Longitudinal growth of long

bones is accomplished by proliferation of chondrocytes at the

growth plates with subsequent bone mineralization as part of

endochondral ossification, although the site of initial tumor

formation (intramedullary or surface lesions) is a matter of

speculation based on the radiographic appearance of the

primary tumor. Most osteosarcomas are defined as high

histologic grade. These tumor originate most often in the

intramedullary space; however, low-grade intramedullary osteo-

sarcomas are seen rarely.(13) Similarly, surface osteosarcomas,

which presumably arise from cells in the periosteum, can occur in

several variants, including parosteal lesions, which are low-grade

osteosarcomas, high-grade surface osteosarcomas, and perios-

teal oteosarcomas that are intermediate in grade.(14) The

treatment of both intramedullary and surface lesions is based

primarily on histologic grade. Little is known about the

comparative biology of these distinctive lesions.(14) Similarly,

axial lesions that arise from bones, which form through

intramembranous ossification, have not been compared in a

comprehensive manner with tumors arising in appendicular

sites. Although osteosarcoma’s bone derivation is likely to be a

critical feature, it is unclear if and how the cellular compartment

from which the tumor arises influences its development and

progression.

Even the cell of origin of osteosarcoma has been the subject of

extensive recent discussion. A number of recent reviews have

challenged the historical belief that osteosarcoma is derived

from osteoblasts. Rather, newer data provide an argument that

the presence of distinct histologic forms of osteosarcoma is a

result of the tumor’s retained potential for pluripotent

differentiation.(1,3,4) Nonetheless, osteosarcomas are categorized

as various histologic subtypes based on their predominant

pattern of differentiation. Histologic subtype does not appear to

influence the clinical behavior of the disease.(1,3,4) Knockout mice

with conditional loss of Rb and p53 function in a preosteoblastic

GORLICK AND KHANNA

Table 2. All Epidemiologic and Genetic Alterations AssociatedWith Osteosarcoma Are Related to Growth/Proliferation or DNA Damage/

Cell Cycle Checkpoint Control

Growth/proliferation-related DNA damage/cell cycle checkpoint control

Epidemiologic associations Association with greater height Radiation exposure

Peak incidence in second decade of life Hereditary retinoblastoma (Rb genetic association)

Earlier peak incidence in females who have

earlier pubertal growth spurts

Li-Fraumeni syndrome (p53 genetic association)

Paget’s disease Rothmund-Thomson syndrome (RECQL4 genetic

association)

Occurrence selectively in large-breed canines Werner syndrome (WRN genetic association)

Parathyroid hormone exposure

Genetic associations MET/HGF Other means of abrogating p53 function (MDM2

amplification, INK4 deletion, COPS3 amplification)EGFR/EGF

HER-2

ErbB-4

MAPK

IGF-1R/IGF Other means of abrogating Rb function (INK4

deletion, CDK4 amplification)BMP

WNT

ß-catenin MYC

VEGFR/VEGF FOS

PDGFR/PDGF SV40

TGFR/TGF

cell develop a tumor that, based on appearance, clinical features,

and gene expression profile, is an osteosarcoma.(15) This suggests

that the cell of origin is this preosteoblast.(15) However, the tumor

that arises in this model system has been noted to express more

primitive lineage markers than preosteoblasts.(16) Although

these markers may be acquired in the transformation process,

it also has been suggested that a rare precursor cell that

expresses markers of both mesenchymal stem cells and

preosteoblasts may be the cell of origin.(16) Targeted disruption

of p53 and Rb in the mesenchymal cells of the murine limb bud

also produces sarcomas.(17) A separate line of evidence comes

from the observation that serially passaged murine mesench-

ymal stem cells will undergo rare spontaneous transformation

resulting in an osteosarcoma-like tumor, suggesting that these

cells are the cell of origin.(18,19) With the existing available data, it

is not possible to define whether osteosarcoma derives from

mesenchymal stem cells or a more differentiated progenitor.

The rarity of osteosarcoma may be related to a small pool of

cells being capable of producing the cancer. Alternatively,

events occurring at the time of maximal longitudinal bone

growth may be necessary for developing osteosarcoma, and this

may be restricting its formation. The events may be related to

aberrations in the differentiation program necessary for

ossification and longitudinal bone growth or may be related

to the need for an external signal initiating cellular proliferation.

These events may trigger the proliferation of a mutated cell, or it

may be proliferation that drives the initial mutation event.

Although limited data exist in this regard, it is not clear that

patients with hereditary retinoblastoma or Li-Fraumeni syn-

drome develop osteosarcomas markedly earlier than individuals

who do not have a germ-line mutation resulting in an

OSTEOSARCOMA

osteosarcoma predisposition despite their markedly higher

incidence of osteosarcoma.(1,4,11) This perhaps is suggestive of

the former scenario, but few data exist to define what initiates

and is necessary for osteosarcoma formation.

A number of mechanistic studies have been performed over

the past 2 years in an attempt to decipher pathways associated

with the pathogenesis of osteosarcoma. Two of the pathways

that have been among the more extensively studied include

Wnt and Notch. The aforementioned observation of sponta-

neous transformation of murine mesenchymal cells into

osteosarcoma has been used to study pathogenesis.(18) A

parallel and functional phenotypic analysis of the parental

mesenchymal stem cells, transformed mesenchymal stem

cells, and resulting osteosarcoma suggested aneuploidization,

translocations, and homozygous loss of the cdkn2 region as

the major mediators of malignant transformation.(18) This

same research group has performed expression profiling of

these cell types and has suggested that downregulation of

Wnt signaling has an important role in osteosarcoma

pathogenesis.(20) When the Wnt pathway was activated using

a GSK3b inhibitor in osteosarcoma cell lines, proliferation was

inhibited, and osteogenic differentiation was observed.(21)

This is further supported by the observation that patients with

osteosarcoma have higher serum levels of Dkk-1, a secreted

inhibitor of the canonical Wnt pathway. Dkk-1 and RANKL also

were noted to be coexpressed by rapidly proliferating

osteosarcoma cells.(22)

In the majority of malignancies, activation of the Wnt pathway

and b-catenin promotes tumorigenesis, in contrast to the

aforementioned line of evidence in osteosarcoma.(23) Indeed,

even in osteosarcoma, a number of experiments have been

Journal of Bone and Mineral Research 685

performed downregulating the Wnt pathway using genetic or

drug-treatment approaches, and in contrast to the prior observa-

tions, enhanced invasion and motility were observed, with the

related clinical observations being an increased frequency of

pulmonary metastases or decreased survival.(24–28) It has been

noted that Wnt inhibitory factor 1 is epigenetically silence in

human osteosarcomas, with targeted disruption in mice accel-

erating osteosarcoma development.(29) With the existing available

data, it appears likely that the Wnt signaling pathway has an

important role in osteosarcoma, but it is unclear whether up- or

downregulation promotes its formation and malignant behavior.

The Notch receptors have an important role in cell fate

decision making and are closely involved in mesenchymal stem

cell differentiation. Since the pathway is involved in numerous

skeletal diseases, it is logical to presume its involvement in

osteosarcoma.(30) Indeed, data from murine models and human

cell lines suggest Notch involvement in osteosarcoma patho-

genesis as well as invasion and metastases. Studies thus far on

the Notch pathway in osteosarcoma are rather limited.(31,32)

Metastases

A defining feature of osteosarcoma is the high rate of metastasis

that results from the primary bone tumor disseminating to

distant secondary sites by a hematogenous route. Despite the

use of multimodality and multiagent systemic cancer therapy

before and after management of the primary tumor, the vast

majority of deaths seen in osteosarcoma patients occur as a

result of metastases. The most common site of metastasis is the

lungs.(1,4) In the era before the use of chemotherapy, most

osteosarcoma patients still had successful management of

the primary tumor (most often through limb amputation).

Nonetheless, over 85% of patients continued to develop

metastases. This suggests that in osteosarcoma patients,

microscopic spread of cancer cells has occurred at the time of

original presentation. The emergence of visible metastases can

occur within months or after a prolonged period of ‘‘dormancy.’’

Dormancy in cancer and in osteosarcoma is poorly under-

stood.(33) First, it is unclear where in the body these dormant

metastatic cells exist. Recent studies support a hypothesis that

the bone marrow may be a site where dormant metastatic cells

reside. Support for this hypothesis in osteosarcoma includes the

identification of osteosarcoma cells in the bone marrow of

patients.(34) Following the rationale of this hypothetical model,

cells would disseminate from the primary tumor early during

tumor formation to the bone marrow. Metastatic cells then

would persist during the period of dormancy in the bonemarrow

and then emerge subsequently and colonize distant secondary

sites at the break in dormancy. The presumed mesenchymal

stem cell origin for sarcomas (discussed earlier) and their ability

to traffick to the bone marrow provide circumstantial support for

this hypothesis. The determinants that result in a break in

dormancy are similarly poorly understood. Experimental data

suggest a link between bursts in angiogenesis and breaks in

dormancy in sarcoma and other cancer models.(35) The causes for

such bursts or the clinical scenarios that may be linked to these

events are not understood.


Once established, metastatic lesions become increasingly

difficult to manage. Unlike most other cancers, the resection of

pulmonary metastasis, when possible, is the common second-

line treatment. Metastectomy is associated with 5-year survival

rates of up to 40% of patients. Unfortunately, subsequent

recurrence in most metastatic patients will be seen and

eventually will require systemic treatment. The eventual

resistance of these pulmonary metastases to currently available

systemic therapy is common. The failure of treatment may be the

result of acquired resistance to chemotherapy and/or to the

ability of metastatic cells to develop ‘‘protection’’ within their

microenvironment in the lung.

The process of metastasis includes tumor cell migration,

invasion, entry into the circulation, and eventual arrest and

extravasation at distant secondary sites, which has been

reviewed extensively.(36,37) Many of the genes that have been

associated with osteosarcoma formation (i.e., oncogenesis) are

likely to contribute to progression and metastases. Unfortu-

nately, as discussed earlier, the underlying genetic complexity

has complicated efforts to identify driving and causal genetic

drivers for metastasis in osteosarcoma despite a number of

biologic motifs (i.e., growth factor signaling paths, angiogenic

phenotype, and mesenchymal stem cell origin) that are

consistently associated with osteosarcoma progression and

may be described as metastasis ‘‘virulence’’ factors, as recently

coined by others.(36,37) The following progression (i.e., virulence)

factors in osteosarcoma have been consistently held across

several investigative platforms and studies(38):

Angiogenesis. The development of an angiogenic phenotype

is a recognized determinant of metastatic cells. In osteosar-

coma, several associations with such an angiogenic phenotype

have been dened. This includes an association with metastatic

risk and primary tumor microvessel density, expression of

angiogenesis-associated growth factors, and the use of inhibi-

tors of angiogenesis in osteosarcoma model systems.(39–41)

Ezrin. The cytoskeleton linker protein ezrin, a member of the

ezrin, radixin, and moesin (ERM) family, has been connected

to the metastatic phenotype in murine, canine, and human

osteosarcoma.(42) It is reasonable that a physical connection

between the actin cytoskeleton and the cell membrane is of

value to a metastatic cell as it engages its microenvironment

in cancer. Studies of ezrin in osteosarcoma have demon-

strated a functional efciency provided by the linkage between

the cell membrane and actin cytoskeleton that is related to

the signal-transduction activity of membrane proteins that

are associated with metastasis.(43–45) The specic mechanisms

associated with ezrin’s role in metastasis is not known; how-

ever, a uniting hypothesis suggests that ezrin is part of a

complex solution used by metastatic cells to deal with the

stresses of the process of metastasis.

Integrins. The integrins are a large family of membrane-

associated receptors that interact primarily with matrix asso-

ciated proteins.(46) Integrin signaling has been suggested to

be a primary mechanism whereby cancer cells interact with

the cellular microenvironment. In osteosarcoma, the expres-

sion of specic integrin family members has been linked to

metastasis.(45)

GORLICK AND KHANNA

OS

Chemokines. Similar to the integrin family of proteins, expres-

sion of chemokines and the chemokine receptors have

been linked to osteosarcoma progression and metastasis in

a number of preclinical and correlative studies.(47,48) Interac-

tions between chemokines and integrin family members

further contribute to the ability of metastatic cells to interact

with their microenvironment and promote metastatic cell

survival.(49)

The insulin-like growth factor 1 (IGF-1) pathway. The IGF-1

pathway has been linked to the development and progres-

sion of many sarcomas, including osteosarcoma.(50) The

growth and development of adult mesenchymal tissues

are largely the result of growth hormone–induced release

of IGF-1 and its interaction with the IGF-1 receptors present

on osteoblasts and other mesenchymal cells. Proliferation

and survival of normal and malignant osteoblasts have been

linked to activation of the IGF-1 pathway.(50) Furthermore, the

roles of the IGF-1 pathway in osteosarcoma include direct

associations with the metastatic phenotype.(51,52) Recent

opportunities to target the IGF-1 receptor have been possible

through humanized and fully human antibodies that target

the IGF-1 receptor and small-molecule inhibitors directed

against IGF-1 receptor kinase. A number of therapeutic anti-

bodies targeting the IGF-1 receptor are in various stages of

preclinical and clinical development in cancers, including

osteosarcoma, and have been associated with single-agent

activity in preclinical models.(53) Additional agents target

downstream components of the IGF-1 receptor pathway,

including PI3 kinase and Akt kinase, which are connected

to progression and metastasis in sarcoma.

c-MET. c-MET is the receptor for hepatocyte growth factor.

The identication and description of c-MET, as an oncogene,

came from studies in a chemically transformed model of

osteosarcoma. Furthermore, preclinical studies in vitro and in

vivo support the role of c-MET signaling in cancer progression

and specically metastasis.(54–56) c-MET has been shown to be

expressed in sarcoma primary tumors and metastatic lung

nodules.(57) It is likely that several metastatic processes are

linked to c-MET signaling, including cell motility, invasion,

proliferation, and survival.(58) Since c-MET is a growth factor

receptor with an intracellular tyrosine kinase activity, the

development of small-molecule inhibitors of c-MET has been

possible. The inhibition of c-MET has been effective in sup-

pressing metastatic phenotype in osteosarcoma cells and

preclinical models.(59)

Mammalian target of rapamycin (mTOR). mTOR is a critical

node in a signaling pathway that connects many growth

factor receptors through intermediaries, including AKT and

MAPK, to the translational machinery of the cell.(60) As a result,

mTOR is able to convert signals that sense the nutritional and

stress status of a cell (i.e., in the cell’s microenvironment) into

specic proteins that can manage the stress.(61) Many of the

known translational targets of mTOR have been connected to

cancer, including c-myc, VEGFR, HIF, and TGFb. The impor-

tance of mTOR in osteosarcoma is also supported by mTOR’s

importance in mesenchymal stem cells.(62) Preclinical studies

with agents that block mTOR have been shown to reduce

TEOSARCOMA

metastases in a murine model of osteosarcoma.(63) Early

human clinical data with mTOR inhibitors support the ther-

apeutic value of this target in many sarcoma histologies.(64)

New Resources and Methods

Much has been written thus far about the complexities in

understanding osteosarcoma, but much of the promise lies in

the new resources that exist for studying the disease. In the

1980s and 1990s, the material that was available to study

osteosarcoma was predominantly cell lines.(7) Not minimizing

the importance of human osteosarcoma cell lines such as the

SaOS-2, HOS, and U-2 OS cell lines (American Tissue Type Culture

Collection, ATTCC), both issues of selection for growth in vitro

and the long duration with which they have been passaged,

among other issues, limit their relevance for studying the human

disease.(7) In the late 1990s, the Children’s Oncology Group

launched a successful national osteosarcoma tissue-banking

effort. At present, over 1000 patients have been enrolled in the

study, with blood and serum available on over 900 individuals,

frozen tumor tissue from over 500, and paraffin-embedded

tumor from over 600. A broad range of assays is planned for

these tissues, but requests for banked material can be made by

all investigators using a Web-based application (https://ccrod.-

cancer.gov/OsteosarcomaSampleRequest/). Applications are

subjected to a peer-review process, but being a part of the

Children’s Oncology Group is not a review criterion. This bank of

tissue, although a tremendously valuable resource, is limited by

the fact that it has been acquired solely through a pediatric

cooperative group and therefore includes only patients up to age

40. Interactionswith other cooperative groups such as the Sarcoma

Alliance for Research Though Collaboration (SARC) ultimately may

permit banking of osteosarcoma tissue from older individuals, but

at present, that tissue resource is not readily available. In addition

to tumor tissue, human osteosarcoma has been serially passaged

in heterotopic sites in immunocompromised mice as another

resource for studying the disease.(65)

New technologies continue to emerge that permit more

comprehensive assessments of tumor tissue. Massively parallel

sequencing is permitting whole-genome sequencing of tumors.

These technologies hold the promise that they may allow an

acquisition of a deeper understanding of the pathogenesis of

osteosarcoma. The prior development of techniques such as

oligonucleotide microarrays were believed previously to hold

significant promise.(7) Unlike sarcomas derived from recurrent

chromosomal translocations, the signatures produced by

expression profiling were exceedingly heterogeneous. Simple

classifications that were possible for the translocation-associated

sarcomas were not possible for osteosarcoma, and these

analyses thus far have produced few tangible results.(66,67)

Perhaps suggesting whole-genome sequencing may be of value

in osteosarcoma is the ability of this approach to identify

consistently mutated pathways in genetically complex malig-

nancies such as lung adenocarcinoma.(68) Whether a consistent

pattern will emerge from whole-genome sequencing analyses of

osteosarcoma awaits completion of these studies.


Preclinical Screening

Even in the absence of understanding osteosarcoma’s patho-

genesis, laboratory studies can be performed that may yield

information that is useful clinically. Identifying biologically based

prognostic factors may not require a fundamental under-

standing of the tumor’s initiation because invariable events are

unlikely to discriminate clinically distinct patient subsets.(7)

Beyond prognostic factors, one can perform empirical preclinical

screening through model systems to identify therapeutically

relevant drugs. This effort is facilitated by the fact that few drugs

are likely to be developed specifically for osteosarcoma. Therefore,

one does not need to identify what is the optimal therapeutic

target in osteosarcomabut rather can focus on screening the drugs

that are likely to be available clinically to determine which of them,

if any, may be effective. Using model systems is much more

efficient, cost-effective, and rapid than performing human clinical

trials, particularly given the rarity of osteosarcoma.

The Pediatric Preclinical Testing Program is one such effort

under way 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 program is able to

quickly screen a large number of agents using in vitro and in vivo

models.(65,69–71) The in vitro models are cell lines, with the in vivo

models typically human patient-derived tumors grown as

heterotopic xenografts in severe combined immunodeficiency

mice. Although osteosarcoma can be grown in orthotopic as well as

heterotopic sites, the heterotopic site permits frequent tumor size

measurements using a caliper, which is feasible and cost-effective.

Preclinical testing potentiallymay predict the activity of new agents

in patients with childhood cancers, allowing identification of active

agents more rapidly. Some believe that preclinical testing needs to

be validated as accurately representing responses in human clinical

trials prior to use 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 because it is more likely to be

predictive than intuitive or random selection. This program has

generated a large amount of data, including characterizations of

the model systems, that has rapidly been published or made

available through Web-based applications (http://home.ccr.can-

cer.gov/oncology/oncogenomics/).(65,69–71)

Canine Models

An important opportunity to extend our understanding of cancer

biology and therapy through preclinical studies is provided by

the natural development of osteosarcoma in pet dogs.(72)

Naturally occurring tumors in dogs and other animals have

clinical and biologic similarities to human cancers that are

difficult to replicate in other model systems. A recently launched

cooperative effort, the National Cancer Institute’s (NCI’s)

Comparative Oncology Trials Consortium (COTC; http://ccr.can-

cer.gov/resources/cop/COTC.asp), provides an infrastructure

and the resources needed to integrate these naturally occurring

cancer models into the development of new human cancer

treatments.(73) The study of cancer biology and therapy in

animals with naturally occurring cancers, referred to as


comparative oncology, is not a novel concept. Indeed, over the

last 30 to 40 years, investigators have used this approach tomake

important contributions to the understanding and practice of

human oncology in fields such as basic tumor biology and

immunology, radiation biology, hyperthermia, and systemic

therapies for a variety of cancers, including osteosarcoma,

lymphoma, melanoma, and others. The parallels between canine

and human osteosarcoma are perhaps the strongest across the

comparative oncology opportunities. Both diseases are char-

acterized by primary tumor growth in the appendicular skeleton

and a high risk for metastasis to the lungs. The canine disease is

indistinguishable from the human disease at the histologic and

gene expression levels. Indeed, both conventional and inves-

tigational treatments for both the primary tumor and the

metastatic disease are associated with similar response features

in both species. The primary differences between the models is

the age of development and the prevalence of disease. In dogs,

osteosarcoma is a disease of older, large breed dogs (i.e., 6 to

12 years of age), whereas osteosarcoma occurs most commonly

in the second decade of life in humans. The incidence of

osteosarcoma in dogs is not known; however, some estimates

suggest well over 10,000 cases annually in the United States. This

high prevalence and the relatively rapid rate of disease

progression (median disease-free interval following surgery

alone is 4 months; with surgery and chemotherapy, 13 months)

provides the opportunity to evaluate novel treatment options in

dogs in a relatively compressed time period. Current studies in

collaboration between the Children’s Oncology Group and the

COTC will attempt to rank the most active agents evaluated in

canine osteosarcoma as part of future consideration in pediatric

osteosarcoma clinical trials.

Osteosarcoma and Its Relationship toNormal Bone Biology

Throughout this review it has been highlighted that osteosar-

coma is not well understood within the context of normal bone

biology. The National Cancer Institute, among other groups, has

sponsored several conferences bringing together sarcoma

researchers with individuals who research mesenchymal stem

cells as well as normal bone physiology attempting to foster

collaborations (http://rarediseases.info.nih.gov/ASP/html/con-

ferences/conferences/sarcoma20040927.html#report). Despite

these conferences, few collaborations of this type are evident

in the literature. The roles of the Wnt and Notch pathways in

osteosarcoma, which are important in mesenchymal stem cell

differentiation and bone formation, have been clarified only to a

limited extent thus far, as has been described previously. Several

researchers explore the tumor-environment interactions for the

more common bone-metastasizing cancers such as breast and

prostate adenocarcinoma.(74–78) It is unclear to what extent these

same mechanisms are operative in osteosarcoma, which arises

from as well as disseminates to bone. This remains a critical

direction for future research efforts. Largely based on the studies

of epithelial cancers that are metastatic to bone, current clinical

and translational studies on the bone microenvironment and

osteosarcoma are limited to the study of the bone osteoclast.

GORLICK AND KHANNA

Unfortunately, the current understanding of the interaction

between the bone osteoclast and osteosarcoma is incomplete.

Such an understanding is necessary for optimal development of

treatment strategies that target the bone osteoclast and bone

osteoclast activation, such as the bisphosphonates and RANK

ligand antibodies in the treatment of osteosarcoma.(79–83)

Conclusions

Although many questions remain unanswered with regard to

understanding the fundamental biology of osteosarcoma, the

availability of a large tissue bank, along with xenograft and

canine model systems, offers considerable promise for the

future. Clinically useful information may be derived from

empirical screening approaches as well as the application of

new laboratory methodologies focused on defining rare but

recurrent genetic drivers of osteosarcoma development and

progression. A critical need for future research efforts will be to

understand osteosarcoma in the context of normal bone

physiology and the environment in which it arises and

progresses. Use of osteosarcoma-related tissue resources that

are available to the entire research community and approaches

to foster collaborations between the sarcoma and bone research

disciplines is encouraged.

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