Incidence

Worldwide

 Latest data showed that in 2021, in the 0 to 14 year age range, the incidence rate of
osteosarcoma in all races and genders is four cases per year per million people. This
number rises to five cases per year per million people (4.6 to 5.6, 95% confidence
interval) for the 0 to 19 year age range (McKeon, 2021).
  The incidence of osteosarcoma has historically been reported as higher in males than in
females, with an incidence rate of 5.4 cases per year per million males and 4 cases per
year per million females, respectively (McKeon, 2021).
 Osteosarcoma accounts for approximately 2.4% of pediatric cancers, making it the eighth
most common childhood malignancy (McKeon, 2021).
 Osteosarcoma is the most common primary bone malignancy in children and adolescents,
representing approximately 400 new cases each year in patients younger than 20 years
(Petriceks, 2019)

Schematic Diagram with Risk Factors and Manifestations 

Biological Risk Factors
1. Height - Children with osteosarcoma are usually tall for their age. This is because
rapid bone growth can contribute to this disease (Mirabello et al., 2011). 
2. Age - The risk of osteosarcoma is highest for those between the ages of 10 and
30, especially during the teenage growth spurt. This suggests there may be a link
between rapid bone growth and risk of tumor formation (Mirabello et al., 2011). 
3. Sex - Osteosarcoma is more common in males than in females. This can be
related to boys being taller on average (Mirabello et al., 2011).
Environmental Risk Factors
1. Chemical Carcinogens
 Foods - Residues of pesticides that can induce tumors may contaminate
foods through their application directly on crops or from other sources of
environmental contamination. Certain unavoidable contaminants in foods,
such as aflatoxin B1 and polychlorinated biphenyls, have been found to be
carcinogenic in long-term toxicological studies. In addition to known
carcinogens that may appear in food as natural constituents, contaminants,
or additives, there are a number of chemicals in food whose carcinogenic
potential has not been adequately assessed but which are suspected
carcinogens because of their known mutagenic activity, i.e., they can
cause heritable alterations in the genetic material of cells (National
Academy of Sciences, 2017). 
 Chemical Agents - Chemical agents such as nickel, nitrites, cadmium,
vinyl chloride, etc. can produce toxic effects by altering DNA structure in
body sites distant from chemical exposure (Hinkle & Cheever, 2017).
 Drugs - Certain drugs such as alkylating agents, immunosuppressant and
estrogen replacement therapy can also produce toxic effects by changing
 the DNA structure in body sites distant from chemical exposure as well
(Hinkle & Cheever, 2017). 
2. Physical Carcinogens
 Radiation - The dose of radiation that can cause cancer is not known and
there is considerable debate surrounding the effect of exposure to low-
dose radiation over a period of time. When cells are exposed to the source
of radiation, it can cause damage to one or both strands of DNA (Hinkle &
Cheever, 2017).
 Viruses - Viruses incorporate themselves in the genetic structure of the
cell, thus altering future generations of cell population (Hinkle & Cheever,
2017).
Genetic Factors
1. Defective RB1 Gene - Inherited RB predisposes primarily young children to both
Retinoblastoma and Osteosarcoma. In this genetic disease, the RB1 gene is
defective, resulting in tumors of the retina. These tumors likely arise because the
pRb encoded by RB1 ordinarily functions in regulating cell proliferation and
differentiation. Without functional pRb, regulation of the G1 to S phase transition
of the cell cycle is lost, leading to unrestricted cell proliferation (Ballatori &
Hinds, 2016).
2. Inactivation of p53 tumor suppressor gene - An autosomal dominant disorder
due to mutations in the p53 tumor suppressor gene, has been found in up to 3% of
children with osteosarcoma. Similar to inherited RB, the mutation results in the
inactivation of a tumor suppressor gene (p53). Functional p53 is responsible for
regulating cell cycle progression in the setting of DNA damage (Prater &
McKeon, 2022; Ballatori & Hinds, 2016). 
3. Defective RECQL, BLM, and WRN genes - These defective genes (RECQL4,
WRN and BLM) encode DNA helicases. These defective DNA helicases lead to
tumors due to disrupted regulation of DNA replication, disrupted regulation of
homologous recombination, and overall genomic instability (Ballatori & Hinds,
2016).
 The transformation of normal cells into cancer cells is brought by different factors. In this
case, it is brought by biologic factors such as age, sex and height. This osteosarcoma often is
highest for those between ages 10-30 males and usually linked to a taller height, all leading to
rapid bone growth experiences. This is because the epiphyseal growth plates of the distal femur
and proximal tibia are responsible for a great deal of the increase in height that occurs during
puberty (Broadhead, et. al., 2011).

On the other hand, the constant consumption of the patient to red and processed meat
contributed as chemical carcinogens under the environmental factors which binds in the
macromolecules in the DNA and RNA proteins. This is because meat processing like adding
nitrates or nitrites and smoking can lead to the formation of potentially cancer-causing
(carcinogenic) chemicals such as N-nitroso-compounds (NOC) and polycyclic aromatic
hydrocarbons (PAH) (Harvard T.H. Chan School of Public Health, 2015). On the other hand,
chemical agents such as nickel, nitrites, cadmium, vinyl chloride, etc. can produce toxic effects
by altering DNA structure in body sites distant from chemical exposure Forming bigger
molecules produces toxic effects that could result in mutations altering the structure of single
genes or chromosomes.

Furthermore, physical carcinogens include radiation wherein when the cells are exposed
to the source of radiation, it can cause damage to one or both strands of DNA. Viruses also  
incorporate themselves in the genetic structure of the cell, thus altering future generations of cell
population (Hinkle & Cheever, 2017).

Under the genetic factors,  inherited RB predisposes primarily young children to both
Retinoblastoma and Osteosarcoma. In this genetic disease, the RB1 gene is defective, resulting in
tumors of the retina. These tumors likely arise because the pRb encoded by RB1 ordinarily
functions in regulating cell proliferation and differentiation. Without functional pRb, regulation
of the G1 to S phase transition of the cell cycle is lost, leading to unrestricted cell proliferation
(Ballatori & Hinds, 2016). Moreover, the same source states that an autosomal dominant disorder
due to mutations in the p53 tumor suppressor gene, has been found in up to 3% of children with
osteosarcoma. Similar to inherited RB, the mutation results in the inactivation of a tumor
suppressor gene (p53). Functional p53 is responsible for regulating cell cycle progression in the
setting of DNA damage and lastly, these defective RECQL4, WRN and BLM genes encode
DNA helicases. These defective DNA helicases lead to tumors due to disrupted regulation of
DNA replication, disrupted regulation of homologous recombination, and overall genomic
instability. 

In cancer development, it starts with the initiation stage in which significantly, it appears
that the acquisition of a single gene mutation is not sufficient to transform normal cells into
cancer cells. There are osteoblastic DNA mutations on extremities of long bones (femur, tibia,
and humerus) near metaphyseal growth plates. Instead, cancerous transformation appears to
require deactivation of anti-oncogenes or the tumor suppressor genes as well as the activation of
many independently mutated genes.

 In the latency stage, a relatively common pathway by which cancer cells gain
autonomous growth is by mutations in genes that control signaling pathways. These signaling
pathways couple growth factor receptors to their nuclear targets, loss of regulator capabilities,
further dysfunction and differentiation happens.

Therefore the role of Tumor Angiogenesis Factor (TAF) takes place in the progression
stage. Even with all the genetic abnormalities described earlier, tumors cannot enlarge unless
angiogenesis occurs and supplies them with the blood vessels necessary for survival. So, there
has been the development and maintenance of independent blood supply. Angiogenesis is
required not only for continued tumor growth but also for metastasis. The molecular basis for the
angiogenic switch is unknown, but it appears to involve increased production of angiogenic
factors or loss of angiogenic inhibitors.

The tumor progression continues as additional mutations occur within cells of the tumor
population. The mutated cells will then have some selective advantage over normal cells as such
there will be rapid growth and division. Cell proliferation of tumors then leads to new clones of
tumor cells with increased growth rate and overcrowding or other properties like survival,
invasion or metastasis.

In the invasion stage, the overcrowding of malignant osteoid tissue can be detected
through a core needle biopsy wherein it shows haphazard arrangement of osteoid tissue and
immature bone. This event could also let malignant cells escape from the primary tumor then
transits within the blood vessels or lymph canal that could colonize and establish metastatic
lesions in different organs which can be clinically manifested by presence of nodules in imaging
tests which could indicate organ failure, and worse, death.

Malignant cancer cells continuously form within the bone wherein the immune system
perceives them as foreign and destroys them, particularly the cell-mediated response. Through
the process of immune surveillance, the immune system recognizes and combat cancer cells
through multiple, interacting cells and actions of the innate, humoral, and cellular components of
the immune system. Tumor-associated antigens (TAAs;b also called tumor cell antigens) are
found on the membranes of many cancer cells. TAAs are processed by antigen-presenting cells
(APCs) such as macrophages and dendritic cells. 

In response to recognizing TAAs as foreign, T-cell lymphocytes release several cytokines

that elicit various immune system actions, including (1) proliferation of cytotoxic (cell-killing) T
lymphocytes capable of direct destruction of cancer cells, (2) induction of cancer cell apoptosis,
and (3) recruitment of additional immune system cells (B-cell lymphocytes that produce
antibodies, natural killer cells, and macrophages) that contribute to the destruction and
degradation of cancer cells. 

Chronically inflamed tissues continue to generate signals that attract leukocytes from the
bloodstream. When leukocytes migrate from the bloodstream into the tissue they amplify the
inflammatory response. By the toxic local inflammatory milieu, it can begin to break down bone
and surrounding soft tissue leading further abnormal proliferation cells. Coussens (2022) said
inflammation is a critical component of tumor progression. This will now lead to the bone
lesions to develop into a bone mass. This could be confirmed by Imaging Tests such as X-ray
and MRI to show an Osteolytic mass on the right proximal end of the tibia of the patient. Tumor
cells will now release tumor-associated proteins leading to elevation of tumor markers.
Moreover, the  bone mass would also cause nerve compression. As the tumor grows it can
compress adjacent nerves and activate local nociceptors on the periosteum resulting in lower
limb pain. The patient may feel chronic pain that interferes with daily activities and difficulty
walking that impairs physical mobility. 

 Moreover, growth factor increases in malignant cells, amplification of proto-oncogenes

or overexpression of growth factors, such as epidermal growth factor (EGF), can lead to
uncontrolled cell proliferation which may manifest as soft tissue swelling/ edema, around the
area of pain due to cell crowding. 

Moreover, the immune system also recognizes and phagocytize invading foreign cells at
a site of affection and then migrate to a nearby peripheral lymphoid organ. They act as antigen-
presenting cells, which directly activate T cells to respond to the microbial antigens. Once
activated, some of the T cells then migrate to the site of infection, where they help other
phagocytic cells, mainly macrophages, destroy the cancer cells. This response would elevate
inflammatory markers such as ESR, CRP and PV. It would also lead to activation of nociceptors
at non-tumorous sites throughout the body manifesting as generalized pain. 

Also, the cytokines involved in the inflammatory chemical milieu stimulates osteoclasts,
the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna
where bone degradation takes place. Stimulating the cells that break down bone results in a net
loss of bone. This bone density causes decreased strength and stability of limbs that may result in
limited ROM, falls, inability to bear weight and increased vulnerability to fractures that may
result in falls and inability to bear weight.  
Core Needle Biopsy
Requesting Physician: Dr. John Guetirrez
Date/Time Ordered: August 1, 2022
Date Received: August 2, 2022
Date Released: August 3, 2022

Definition: A type of biopsy in which a large hollow needle is inserted through the skin to the
site of an abnormal growth to collect and remove a sample of cells for analysis. This procedure
uses an automated needle, which obtains one sample of tissue at a time and is re-inserted several
times (National Cancer Institute, 2022)

Purpose: It is needed after the physical exam, laboratory analysis, and diagnostic imaging
confirm the presence of a lesion consistent with osteosarcoma to determine the definitive
diagnosis, grading, and histological subtyping. (Prater and McKeon, 2022)

 Results:

Histological slide from core needle biopsy of the right proximal tibia. Note the woven, haphazard
arrangement of osteoid tissue and immature bone. Bottom left-hand corner shows a region of
darker, mature lamellar bone. Upper left-hand corner shows a region of immature, ossifying
tissue, more organized than surrounding light-pink osteoid material. Magnification = ×80.

Analysis:

The histological image shows spindle-like, pleomorphic bone-forming cells,

characteristic of a malignant neoplasm. Loosely woven pink osteoid tissue occupies most of the
image, suggesting that these are osteoid-producing cells since one can tell osteoid tissue from
normal bone, by identifying the lighter, less-organized immature proteinaceous material
(Petriceks et. al., 2019). As a whole, the image shows haphazard arrangements of mature and
immature bony material which is a grade 2 according to histologic involvement in which cells
are more abnormal (moderate dysplasia) and moderately differentiated (Hinkle and Chiver,
2014). Also, in combination with the radiograph from X-ray and MRI, this is an osteosarcoma
which has been rapidly producing irregular osteoid tissue.

Nursing Responsibilities

Nursing Responsibilities Rationale

1. Verify doctor’s order.          To prevent error

2. Identify the patient by asking him to state  To prevent error

his name and checking the identification
wristband.  

3. Inform the patient and significant others  To provide information, alleviate

of the procedure, including its nature, anxiety, and promote cooperation
purpose, and time required to perform the
procedure.       

4. Describe what discomfort to expect  To condition the patient to

during the procedure. upcoming procedures.

5. Answer any questions and address any  To provide information, alleviate

concerns voiced by the patient and their anxiety, and show support
significant others.  

6. Ensure the patient can remain still and  To avoid possible injury
follow directions for the specified biopsy.

7. Direct the patient to take slow, deep  To minimize the feeling of pain
breaths when the biopsy needle is
injected.

8. Inform the significant others that the  The requesting physician will
results will be interpreted by the discuss the findings to the patient
physician. 

9. Monitor the patient’s vital signs  To monitor physiological stability.

regularly.

10. Instruct the patient and significant others  To provide early interventions
in the care and assessment of the site.
 Immediately report  any redness, edema,
bleeding, pain at the biopsy site, or any
chills or fever.

11. Document interventions rendered  To ensure that interventions are

rendered.

Famotidine

Time and Date ordered: 

a. Generic Name: Famotidine
 b. Brand Name: Pepcid
c.  Classification
Therapeutic class: Anti-ulcer agents
Pharmacologic class: H2 Receptor Antagonist
d. Dosage and Frequency: Famotidine 20 mg 1 tab 30 mins prior to chemo
e. Route: Oral
f. Mechanism of Action: Competitively inhibits action of histamine on the H2-receptor
sites of parietal cells, decreasing gastric secretion.
g. Desired Effect: To prevent and control heartburn, nausea and chest pains associated
with chemotherapy-induced gastric mucosal injury.
h. Nursing Responsibilities: 

Nursing Responsibilities Rationale

1. Verify doctor’s order.   To facilitate smooth flow of carrying

out medication orders and to avoid
errors

2. Perform hand hygiene.  To avoid contaminating the drug.

3. Observe the 12 rights of medication.  To ensure that the patient receives

Perform the three checks prior to drug the proper medication and promotes
administration.  best patient outcomes.

4. Introduce yourself to the patient or  To help establish rapport and gain

significant others and explain the cooperation and to alleviate anxiety
procedure. Give opportunity to ask
questions

5. Explain the medication to be given and  To facilitate an understanding

its purpose. Give the patient an regarding drug treatment.
opportunity to ask questions. 

6. Give a glass of water to the patient to  For easy swallowing of the medicine
drink the medicine with

7. Do not leave the patient. Make sure the  To make sure that the drug ordered
medicine is taken is taken by the patient

8. Encourage water intake.  To potentiate absorption and

dissolution of the medication.

9. Increase fiber and fluid intake  To prevent constipation

 10. Document the medication given  Serves as a legal basis. (anything
that is not documented is considered
not done)

11. Monitor and document patient  To provide timely interventions. 

responses to the drug and the side
effects.

ADDITIONAL READINGS

S. Siddiqui et al. / J Oncol Sci 5 (2019)

Pathophysiology

The origin of OS is the mesenchymal cells and their histological hallmark is based on the production of
malignant osteoid. Inactivation of P53 and RB pathways is a major event in OS genesis.22 Once mutation
of DNA occurs, it activates the oncogene which leads to a deactivation of the suppressor gene and
produces malignant osteoblasts that lead to a proliferation of abnormal osteoblasts. This causes the
formation of malignant osteoid tissue. The osteoid tissue results in uncontrolled growth of the tumor
that causes overgrowth of malignant osteoid tissue which causes suppression of red bone marrow.
Suppression of bone marrow causes decrease in blood cells which further weaken the immune system
and make susceptible the body to severe infections as the body is unable to produce leukocytes against
pathogens. A decrease in RBC leads to anemia, fatigue, weakness, and a decrease in platelets leads to
bleeding and brushing. The overgrowth and crowding of osteoid cells generates pressure inside the
bone which is responsible for the pain and pathological fractures.

The Molecular Pathogenesis of Osteosarcoma: A Review

Matthew L. Broadhead,1 Jonathan C. M. Clark,1 Damian E. Myers,1 Crispin R.

Dass,2 and Peter F. M. Choong

2. Pathogenesis

2.1. Bone Growth and Tumorigenesis

Osteosarcoma has a predilection for developing in rapidly growing bone. A number of studies
have established a correlation between the rapid bone growth experienced during puberty and
osteosarcoma development [4, 5]. Fifty-six percent of all osteosarcomas present around the knee
[2]. The epiphyseal growth plates of the distal femur and proximal tibia are responsible for a
great deal of the increase in height that occurs during puberty. Additionally, the peak age of
osteosarcoma development is slightly earlier for females, an observation that may be explained
by the relatively earlier growth spurt experienced by girls [6]. There is a male:female ratio of
1.5 : 1 for osteosarcoma, and patients affected by the disease are taller compared to the normal
population of the same age group [7]. Patients affected by Paget’s disease, a disorder
characterised by both excessive bone formation and breakdown, also have a higher incidence of
osteosarcoma [2].

2.2. Environmental Factors

Physical, chemical, and biological agents have been suggested as carcinogens for osteosarcoma.
Among these, the role of ultraviolet and ionising radiation is the best established. The initial
pathogenic link between radiation exposure and osteosarcoma was noted in female radium dial
workers who applied radium to watch faces to make them luminescent [8]. However, radiation
exposure is implicated in only 2% of cases of osteosarcoma [9] and is not thought to play a major
role in paediatric disease. An interval of 10–20 years between exposure and osteosarcoma
formation has been observed [10]. When radiotherapy is used in children as a treatment agent for
a solid tumour, 5.4% develop a secondary neoplasm, and 25% of these are sarcomas [11].
The chemical agents linked to osteosarcoma formation include methylcholanthrene and
chromium salts [12], beryllium oxide [13], zinc beryllium silicate [14], asbestos, and aniline dyes
[15]. Previously, a viral origin had been suggested for osteosarcoma. This stemmed from the
detection of simian virus 40 (SV40) in osteosarcoma cells. However, the presence of SV40 in
these cells was later concluded to be the result of presence of SV40 viral units as contamination
in the polio-virus vaccine that these patients had received [16, 17]. Studies evaluating the role of
SV40 in the pathogenesis of mesothelioma have suggested that detection of SV40 in human
cancers may in fact be due to laboratory contamination by plasmids containing SV40 sequences
[18, 19].

2.3. Chromosomal Abnormalities

A number of chromosomal and genetic syndromes have been linked to osteosarcoma.

Osteosarcoma has been reported in patients with Bloom syndrome, Rothmund-Thompson
syndrome, Werner syndrome, Li-Fraumeni syndrome, and hereditary retinoblastoma [15].
Bloom, Rothmund-Thompson, and Werner [20] syndromes are characterised by genetic defects
in the RecQ helicase family. DNA-helicases are responsible for separation of double-stranded
DNA prior to replication [21, 22]. Mutations in these genes confer a higher risk of multiple
malignancies.

A recent study of pretherapeutic biopsy specimens has identified amplifications of chromosomes

6p21, 8q24, and 12q14, as well as loss of heterozygosity of 10q21.1, as being among the most
common genomic alterations in osteosarcoma. Furthermore, it was concluded that patients
carrying these alleles had a poorer prognosis [23]. Numerical chromosomal abnormalities
associated with osteosarcoma include loss of chromosomes 9, 10, 13, and 17 as well as gain of
chromosome 1 [24].

2.4. Tumour Suppressor Gene Dysfunction

When human cells are exposed to environmental insults, such as those discussed above, somatic
DNA may be damaged. Such DNA damage may not necessarily give rise to a malignant cell line,
as there are a number of tumour-suppressor mechanisms in place. These mechanisms may either
repair the DNA damage or induce apoptosis of these cells. The p53 and retinoblastoma (Rb)
genes are well-known tumour-suppressor genes. However, tumour suppressor genes may
themselves become mutated, resulting in the loss of their protective function. As a result,
additional somatic mutations may accumulate, giving rise to a cell line that replicates without
restraint. Mutations in both the p53 and Rb genes have been proven to be involved in
osteosarcoma pathogenesis [6].

The p53 gene is mutated in 50% of all cancers and 22% of osteosarcomas [24]. DNA damage
results in phosphorylation of p53, which is constitutively inhibited by Mdm2. Phosphorylation
allows p53 dissociation from Mdm2. p53 exerts its tumour-suppressor effects via the activation
of proapoptotic Bax and p21. The latter binds and inactivates G1/S-Cdk and S-Cdk complexes,
causing arrest of the cell cycle in G1 [25].

Recently, p53 mutations have been shown to result in impaired DNA repair mechanisms and
disrupted antiangiogenesis activity [26]. For osteosarcoma, the prototypical condition of p53
mutation is Li-Fraumeni syndrome. This syndrome is characterised by an autosomal dominant
mutation of p53 leading to the development of multiple cancers including osteosarcoma [27]. Li-
Fraumeni syndrome and germ-line mutations of p53 in osteosarcomas are rare, however [28],
and in many osteosarcoma cell lines, a mutation in the first intron of the p53 gene occurs [29]
though other point mutations have also been reported [30].

While p53 has been implicated in the oncogenesis of osteosarcoma, it is unclear whether p53
mutation or loss may affect tumour behaviour. Using the p53-null SaOS-2 osteosarcoma cell
line, Ganjavi et al. [31] showed that adenoviral-mediated gene transfer of wild-type p53 resulted
in reduced cell viability and increased sensitivity to chemotherapeutic agents. A recent study
published by Hu et al. [32] showed that p53 expression was higher in low Rosen grade
osteosarcomas (Rosen grade 1: <50% necrosis; grade 2: 50%–90% necrosis; grade 3: >90%
necrosis; grade 4: 100% necrosis; grade 1 + 2 = low-grade; grade 3 + 4 = high grade). p53
expression correlated with reduced metastatic disease and improved survival for these patients.
p53 mutation has also been shown to be more common in high-grade conventional
osteosarcomas versus low grade central osteosarcomas [33]. However, other studies differ such
as that of Lonardo et al. [34], which found no relationship between p53 and histological grade.
Univariate analysis performed by Park et al. [35] showed no correlation between survival and the
p53 protein, while coexpression of p53 and P-glycoprotein was associated with a poorer
prognosis.

In addition to p53, the Rb tumour suppressor has also been implicated in the tumorigenesis of
osteosarcoma. The Rb gene is critical to cell-cycle control, and inherited mutation of the Rb gene
causes retinoblastoma syndrome, a condition that predisposes a patient to multiple malignancies
including osteosarcoma. The Rb protein regulates the cell cycle by binding the transcription
factor E2F. E2F is held inactive by Rb until the CDK4/cyclin D complex phosphorylates Rb.
Mutations of Rb allow for the continuous cycling of cells [25].

Both germ-line and somatic mutations of Rb confer an increased risk of osteosarcoma. Loss of
the Rb gene may even explain the familial risk of osteosarcoma [36]. However, it has yet to be
determined whether Rb gene loss or suppression gives rise to more aggressive tumours with
poorer prognosis. Loss of heterozygosity for Rb has been reported to confer both an improved
and poorer prognosis for patients [37–40]. In terms of response to chemotherapeutic treatment,
Iida et al. [41] showed that the SaOS-2 osteosarcoma cell line, lacking active Rb, was less
sensitive to the growth-suppressing effect of methotrexate compared to cell lines with wild-type
Rb gene. Further studies are warranted to investigate the role of Rb on chemosensitivity of
osteosarcoma cells.

2.5. Transcription Factors

Transcription is the process of forming single-stranded messenger RNA (mRNA) sequences
from double-stranded DNA. Transcription factors facilitate binding of promoter sequences for
specific genes to initiate the process. While transcription is usually tightly regulated,
deregulation may occur in osteosarcoma, as with other cancers. Excess production of
transcription factors, or the production of a new overactive transcription factor, may result from
gene rearrangement.

The activator protein 1 complex (AP-1) is a regulator of transcription that controls cell
proliferation, differentiation, and bone metabolism. AP-1 is comprised of Fos and Jun proteins,
products of the c-fos and c-jun proto-oncogenes, respectively. Fos and Jun are found to be
significantly upregulated in high-grade osteosarcomas compared with benign osteoblastic lesions
and low-grade osteosarcomas [42, 43] and are associated with the propensity to develop
metastases [44]. Fos and Jun double-transgenic mice are found to develop osteosarcomas with a
higher frequency than c-Fos only transgenic mice [45]. Most recently, Leaner et al. [46] showed
that inhibition of AP-1-mediated transcription caused reduced migration, invasion, and
metastasis in a murine model of osteosarcoma. Another approach has been to target the Jun
component of AP-1. The DNA enzyme Dz13 cleaves human c-Jun mRNA and is capable of
inhibiting osteosarcoma growth and progression in a clinically relevant murine model when
delivered by nanoparticle vector [47].

Myc is a transcription factor that acts in the nucleus to stimulate cell growth and division. Myc
amplification has been implicated in osteosarcoma pathogenesis and resistance to
chemotherapeutics. Overexpression of Myc in bone marrow stromal cells leads to osteosarcoma
development and loss of adipogenesis [48]. Myc is amplified in U2OS osteosarcoma cell-line
variants with the highest resistance to doxorubicin, and gain of Myc was found in SaOS-2
methotrexate-resistant variants [49]. Additionally, Myc has been examined as a therapeutic target
for osteosarcoma. Downregulation of Myc enhanced the therapeutic activity of methotrexate
against osteosarcoma cells [50]. Adenovirus-mediated transfection with the antisense Myc
fragment led to cell-cycle arrest and enhanced apoptosis in the MG-63 osteosarcoma cell line
[51]. Using a conditional transgenic mouse model, Arvanitis et al. [52] showed that Myc
inactivation caused proliferative arrest and promoted differentiation in osteosarcoma.
Additionally, using positron emission tomography (PET), these tumours exhibited reduced
metabolic activity as demonstrated by reduced uptake of [18F]-fluorodeoxyglucose ([18F]-FDG).

2.6. Growth Factors

Osteosarcoma cells produce a range of growth factors that exert autocrine and paracrine effects.
Dysregulated expression of growth factors such as transforming growth factor (TGF), insulin-
like growth factor (IGF), and connective tissue growth factor (CTGF) leads to the accelerated
proliferation of cells. Growth factor receptors may be overexpressed and constitutively activated.
Signal transduction associated with these receptors may also be overactivated.

Transforming growth factor beta (TGF-β) proteins are a large family of dimeric proteins secreted
by cells. Like many other growth factors, they influence a wide variety of cell process such as
differentiation, proliferation, apoptosis, and matrix production. Bone morphogenic proteins
(BMPs) make up a large component of the TGF-β family. High-grade osteosarcomas are found
to express TGF-β1 in significantly higher amounts than low-grade osteosarcomas [53]. Navid et
al. [54] examined the autocrine role of TGF-β on two osteosarcoma cell lines, demonstrating a
30%–50% reduction in growth when osteosarcoma cells were cultured in the presence of TGF-β-
blocking antibody. Smad activation was implicated downstream of TGF-β with an inability to
phosphorylate the Rb protein. Most recently, Hu et al. [55, 56] have shown an association
between increased susceptibility and metastasis of osteosarcoma with TGFR1 variants,
TGFBR1*6A, and Int7G24A.

IGF (insulin-like growth factor)-I and IGF-II are growth factors that are often overexpressed by
osteosarcomas. These ligands bind corresponding receptors such as IGF-1R, leading to activation
of the PI3K and MAPK transduction pathways. This, then, supports cell proliferation and
inhibition of apoptosis [57]. The growth-stimulating effect of IGF has been targeted for
osteosarcoma. Lentivirus-mediated shRNA targeting IGF-R1 enhanced the chemosensitivity of
osteosarcoma cells to docetaxel and cisplatin [58]. The use of monoclonal antibodies targeting
IGF-R1 was also effective in enhancing antitumour response [59, 60].

Connective tissue growth factor (CTGF) is related to a number of proteins in the CCN family
(CTGF/Cyr61/Cef10/NOVH). This protein family appears to act via integrin signalling pathways
[61] and, like TGF-β, has a diverse range of functions including adhesion, migration,
proliferation, survival, angiogenesis, and differentiation. Nishida et al. [62] showed that CTGF is
a potent stimulator for the proliferation of SaOS-2 cells, leading to increased expression of type I
collagen, alkaline phosphatase, osteopontin, and osteocalcin, markers for bone cell
differentiation and maturation. A related protein, CCN3, was found to be overexpressed in
osteosarcoma and associated with a worse prognosis [63].

Parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), and the receptor
(PTHR1) have been implicated in the progression and metastasis of osteosarcoma. PTHrP was
discovered as the humoral factor associated with tumour metastasis and hypercalcaemia [64].
The role of PTHrP and PTHR1 in osteoclast signalling will be discussed later. In terms of direct
effects on osteosarcoma cells, when HOS osteosarcoma cells were overexpressed with PTHR1,
increased proliferation, motility, and invasion through Matrigel were observed [65]. Gagiannis et
al. [66] recently showed that PTHrP confers chemoresistance in osteosarcoma by blocking
signalling via p53, death-receptor and mitochondrial pathways of apoptosis. PTHrP
downregulated expression of proapoptotic Bax and PUMA and upregulated antiapoptotic Bcl-2
and Bcl-xl. Berdiaki et al. [67], using MG-63 and SaOS-2 osteosarcoma cell lines, showed that
PTH peptides enhanced osteosarcoma cell migration through the regulation of hyaluron
metabolism. However, a previous study showed that overexpression of PTHrP in a murine
osteoblastic osteosarcoma cell line reduced cell proliferation by 80% [68]. Further studies are
required to determine the prognostic significance of PTH/PTHrP/PTHR1 signalling in
osteosarcoma.

2.7. Osteosarcoma Cell Proliferation, Apoptosis, and Anchorage-Independent Growth

Cancer cells are relatively resistant to apoptosis, and this ability to avoid elimination contributes
to the ability of osteosarcoma cells to proliferate without restriction. Apoptosis consists of
initiation and execution phases. During initiation, enzymes responsible for the cleavage of vital
cellular proteins, known as caspases, are activated. Execution refers to the actual process of
hydrolysis performed by activated caspases. Both extrinsic and intrinsic pathways regulate the
initiation phase. The extrinsic pathway is a death receptor-initiated pathway, while the intrinsic
pathway relies on increased mitochondrial permeability. Both proapoptotic and antiapoptotic
factors interact with these pathways, and these have been discussed in a previous review [69].

Anoikis is a form of apoptosis that is induced when cells are no longer attached to a basement
membrane or matrix. This is of particular interest in osteosarcoma given the propensity of
osteosarcoma cells to detach from matrix components and to metastasise. Osteosarcoma cells are
resistant to anoikis and proliferate despite deranged cell-cell and cell-matrix attachments. This
resistance to anoikis is termed anchorage-independent growth (AIG).

The pathways causing anoikis disruption and leading to anchorage-independent growth are
complex. They involve interactions between integrin signalling, Rho GTPases, PI3 kinase, and
PKB/Akt activation, along with many key components of the intrinsic and extrinsic apoptosis
pathways (Figure 1). For example, when normal cells adhere to surrounding matrix via integrin-
fibronectin binding, the Bcl-2 inhibitor Bit1 is suppressed allowing Bcl-2 to prevent apoptosis
via the intrinsic pathway [70]. Another pathway involves the exchange of integrin subunits
resulting in the production of abnormal integrins, such as αvβ6, which can upregulate PI3 kinase
function [71]. PI3 kinase can then activate PKB/Akt which inhibits the proapoptotic factor Bad,
leading to cancer cell survival [72]. Rho GTPases such as Rac1 and Cdc42 can also upregulate
PI3 kinase with similar consequences [73]. Increased epidermal growth factor-receptor
(EGF/EGFR) binding with subsequent extracellular signal-regulated kinase (Erk)/microtubule-
associated protein kinase (MAPK) signalling leading to inhibition of Bim has also been
described [74]. This suppresses cell death, as Bim would normally act to increase mitochondrial
outer membrane permeability allowing release of cytochrome c and then the activation of
executioner caspases.

2.8. Tumour Angiogenesis

Tumour angiogenesis is essential for sustained osteosarcoma growth and metastasis. Without a
supporting vasculature, osteosarcoma cells would be unable to obtain the nutrients and oxygen
necessary for proliferation. Metastasis to the lungs and bone, the most common sites for
osteosarcoma spread, also relies on the formation and maintenance of blood vessels. Radiation
therapies, while compromising tumour cells, also destroy the vascular component of tumours and
block the supply of nutrients. So, radio- and chemotherapies act by these dual actions. This
aspect is discussed below.

A balance between pro-angiogenic and antiangiogenic factors regulates angiogenesis, and this
balance is tipped towards the favour of neovascularisation by tissue hypoxia, acidosis, oncogene
activation, and loss of tumour suppressor gene function. A hypoxic and acidotic
microenvironment exists around proliferating osteosarcoma cells, and these conditions stimulate
deubiquitination of von Hippel Lindau protein. Von Hippel Lindau protein releases hypoxia-
inducible factor-1α (HIF-1α), allows HIF-1α to bind to the promoter region of the vascular
endothelial growth factor (VEGF) gene [75], and upregulats it. TGF-α, and fibroblast growth
factor (FGF) may also upregulate VEGF [76].

VEGF is the best-characterised pro-angiogenic factor, and it stimulates the processes of

endothelial cell proliferation, migration, and blood vessel maturation. A number of different
VEGF molecules exist (VEGF-A through to VEGF-E), and these proteins bind to VEGF
receptors (VEGFR1-3) [77]. VEGF-A has the broadest angiogenic effect. Upon VEGF-A
binding to VEGFR2, a number of divergent signalling pathways are initiated [77]. Nitric oxide
(NO) is released by endothelial cells, leading to vasodilation and increased vascular permeability
[78]. Endothelial cell proliferation and cycling are stimulated via phospholipase Cγ (PLCγ),
protein kinase C (PKC), and the c-Raf-MEK-MAPK cascades [77]. Rearrangement of the actin
cytoskeleton, necessary for endothelial cell migration occurs via phosphorylation of T cell-
specific adapter (TSAd) and interaction with Src, another protein kinase [79]. The net result of
all these changes is the formation of an immature, irregular, and leaky vascular network.

The immature and inefficient nature of the vessels so produced facilitates feedback loops for
further vessel formation. Upregulation of HIF-1α and VEGF [80] again occurs as the leaky
vasculature is unable to meet the metabolic demands of the proliferating osteosarcoma cells.
Additionally, VEGF upregulates matrix metalloproteinase (MMP) and plasmin activity [81].
These proteases break down extracellular matrix, which releases any VEGF combined with
heparin proteoglycan in the matrix. VEGF also induces antiapoptotic factors Bcl-2, and survivin,
ensuring ongoing endothelial proliferation [82]. In addition to VEGF, the proliferating tumour
cells release a number of other pro-angiogenic factors. These include FGF, platelet-derived
growth factor (PDGF), angiopoietin1 (Ang1), and ephrin-B2 [83, 84].

While it is known that osteosarcoma is a relatively vascular tumour, the prognostic significance
of this is yet to be determined. There have been studies suggesting both a correlation [85, 86] and
lack of association [87] between VEGF expression and osteosarcoma microvascular density and
metastases at diagnosis. This may relate to a greater tumour dependence on functionally mature
vessels. One study that demonstrated a survival advantage associated with increased
osteosarcoma microvascular density [88] attributed this advantage to improved tissue penetration
by chemotherapeutic agents.

As previously mentioned, angiogenesis is regulated by the balance between pro-angiogenic and

antiangiogenic factors. Antiangiogenic proteins such as thrombospondin 1, TGF-β [89], troponin
I, pigment epithelial-derived factor (PEDF) [90], and reversion-inducing cysteine rich protein
with Kazal motifs (RECK) [91] are downregulated in osteosarcoma. These antiangiogenic
molecules are particularly important for embryogenesis and physiological processes such as
wound healing and menstruation; however, they also play a protective mechanism against
osteosarcoma progression. For example, troponin I and PEDF are expressed predominately
within the avascular zones of the cartilaginous growth plate [92, 93] and are likely to contribute
to growth plate resistance to osteosarcoma invasion from a typical metaphyseal location. In
addition to inhibiting angiogenesis, PEDF exerts direct effects on osteosarcoma cells. Ek et al.
[94, 95] have demonstrated apoptosis induction in osteosarcoma cell lines treated with PEDF.
Also, in a murine model of orthotopic osteosarcoma, tumour volume was reduced by PEDF,
which was associated with reduced microvascular density. There was decreased tumour
metastases and reduced size of metastatic tumours in lung.

2.9. Cell Adhesion and Migration

Osteosarcoma is a highly metastatic tumour, and pulmonary metatases are the most common
cause of death. The metastatic sequence involves the detachment of osteosarcoma cells from the
primary tumour, adhesion to the extracellular matrix, local migration and invasion through
stromal tissue, intravasation, and extravasation. The ability of osteosarcoma cells to metastasise
by such a pathway relies on complex cell-cell and cell-matrix interactions.

The extracellular matrix is composed of various protein fibrils and growth factors. The proteins
include fibronectin, collagens, proteoglycans, and laminins. Osteosarcoma cells may also
produce matrix proteins. The extracellular matrix provides a developing tumour with a
supporting scaffold and facilitates blood vessel formation. Osteosarcoma cells adhere to matrix
components via cell-surface receptors. These receptors are more than just a physical point of
attachment; they also provide a link between matrix proteins and the cytoskeleton. The principle
receptor proteins are the integrins, which bind to the matrix protein fibronectin. There are 24
different integrin heterodimer molecules consisting of different α and β subunits [96].

The integrins also play a role in cell signaling, particularly in pathways critical to cell migration.
Integrin-binding proteins such as talin become associated with the cytoplasmic domain and act,
via adaptor proteins such as vinculin, paxillin, and α-actin, for the upregulation of protein kinases
[97]. The key enzymes involved here are focal adhesion kinase (FAK), protein kinase C (PKC),
PI3 kinase, Src, and the RhoA GTPases.

The relative activities of these enzymes underlie conformational changes in cell architecture. For
example, there is a shifting balance between two of the RhoA GTPases: Rac1 and RhoA. High
Rac1 expression suppresses RhoA and induces the formation of membrane ruffles. These
membrane changes facilitate cell spreading and migration [98]. Conversely, high RhoA with low
Rac1 leads to membrane retraction. These two processes are coordinated such that in cell
migration, the leading edge of the cell is demonstrating actin polymerisation and lamellipoedia,
while the trailing edge is undergoing actin disassembly. Inhibition of RhoA pathways has been
shown to reduce osteosarcoma cell migration and invasion [99].

In general, cells migrate towards ligand-dense matrix and towards more rigid matrix [100],
indicating a constant intracellular response to extracellular adhesion and tension. Tumour stroma
is more rigid than normal connective tissue matrix, and this generates integrin clustering,
activation of intracellular signalling pathways, decreases cell-to-cell contacts, and stimulates
tumour growth [101].
The ezrin protein also has a role in cell-cell interactions, signal transduction, linkage between
actin filaments, and cell membrane receptors such as CD44, which binds hyaluronan in the
extracellular matrix. When ezrin is overexpressed, it is associated with an increase in metastasis
[102]. Increased ezrin expression in paediatric osteosarcoma patients is associated with reduced
disease-free intervals, and downregulation of ezrin expression in a mouse model of human
osteosarcoma has been shown to reduce pulmonary metastasis [103].

2.10. Tumor Invasion

Invasion of the surrounding tissues by osteosarcoma also involves degradation of the

extracellular matrix. Matrix metalloproteinases (MMPs) are principally involved in the
breakdown of the extracellular matrix, although roles in tumour angiogenesis have also been
established.
MMPs are a family of zinc-dependent endopeptidases that are involved in a range of
physiological processes including inflammation, wound healing, embryogenesis, and fracture
healing. In normal tissues, MMPs are regulated by natural inhibitors such as tissue inhibitors of
MMPs (TIMPs), RECK, and α2 macroglobulin [104]. In the setting of osteosarcoma, MMPs
break down extracellular collagens, facilitating both tumour and endothelial cell invasion. MMPs
may be designated as gelatinases, collagenasesm, or stromeolysins. Gelatinases break down
denatured collagens and type IV collagen. Collagenases break down type I, type II, and type III
collagen, and stromeolysins break down proteoglycan (found in articular cartilage), type III, type
IV (in basement membranes), and type V collagen, as well as casein and fibronectin [105].

In addition to clearing a pathway for invading osteosarcoma cells, the role of MMPs in
angiogenesis has already been mentioned. Remodelling of vessel walls by MMPs gives rise to a
thin and leaky vascular network that allows passage of tumour cells into the bloodstream [106].
Furthermore, MMP-9 releases VEGF stored within the extracellular matrix [107], and VEGF is
able to upregulate MMP-2 [108]. The specific importance of the gelatinases MMP-2 and MMP-9
to tumour progression has been delineated in an in vivo study, where combined MMP-2/MMP-9
deficiency in mice significantly impaired tumour angiogenesis and invasion [109].

The urokinase plasminogen activator (uPA) system is the other key regulator of osteosarcoma
invasion, which interacts with MMPs. The ligand uPA binds to its receptor uPAR to become
active. Once activated, uPA cleaves plasminogen to plasmin. Plasmin breaks down the
extracellular matrix but also activates pro-MMPs. A cascade of activation is hence established
[110, 111]. The role of the uPA-uPAR system is well established in osteosarcoma pathogenesis.
An inverse relationship between uPA levels and survival time has been demonstrated [112].
Downregulation of uPAR in an in vivo osteosarcoma model resulted in reduced primary tumour
growth and fewer metastases [113].

2.11. Osteoclast Function

Osteosarcoma invasion of bone relies on interactions between the bone matrix, osteosarcoma
cells, osteoblasts, and osteoclasts (Figure 2). Osteoclasts are the principle bone-resorbing cells,
and the substantial osteolysis exhibited by some osteosarcomas is the direct result of increased
osteoclastic activity. During the initial stages of osteosarcoma invasion, growth factors such as
TGF-β are released from the degraded bone matrix and act on osteosarcoma cells, stimulating the
release of PTHrP, interleukin-6 (IL-6) and interleukin-11 (IL-11) [114, 115]. These cytokines
then stimulate osteoclasts, facilitating further invasion and release of proresorptive cytokines.

Osteoblasts are, in fact, mediators in this process of bone resorption. Osteosarcoma cells release
endothelin-1 (ET-1), VEGF, and PDGF in response to the hypoxic and acidotic conditions.
These factors have predominantly osteoblast-stimulatory functions [116, 117]. PTHrP and IL-11
also act on osteoblasts, stimulating increased expression of receptor activator of nuclear
factor κB ligand (RANKL). RANKL is a key mediator of osteoclast differentiation and activity,
and osteosarcoma cells have been noted to produce RANKL independently [118].

RANKL activates osteoclasts through binding to RANK on the osteoclast surface. RANK
expression is under control of cytokines IL-1, IL-6, IL-8, tumour necrosis factor-α (TNF-α),
PTHrP, and TGF-α [119]. Receptor-ligand binding initiates a cascade of events through binding
of TRAF-6, leading to activation of both NFκB and MAPK pathways, with a resulting increase
in nuclear factor of activated T-cells (NFATc1) activity. RANK/RANKL also activates the c-Fos
component of AP-1, resulting in additional NFATc1 upregulation. NFATc1 is thus a common
end-point for effecting transcription of genes involved in osteoclast activity and maturation
[120].

Activated osteoclasts release proteases to resorb the nonmineralised components of bone.

Cathepsin K (Cat K) is a cysteine protease selectively produced by osteoclasts for breakdown of
collagen I, osteopontin, and osteonectin [121]. Cat K is also produced by some cancer cells to aid
invasion [122]. This protease is essential for osteoclast function in normal bone remodelling and
also in pathological states of osteolysis. For patients with high-grade metastatic osteosarcoma,
low Cat K levels at the time of diagnosis confers a better prognosis [123].

c-Src is a nonreceptor tyrosine kinase present within osteoclasts [124] and is involved in
pathways regulating cell growth, survival, and migration [125]. Osteoclast survival occurs
through c-Src mediating phosphatidylinositol 3-kinase and TRAF-6 interaction, with resulting
Akt/mTOR (mammalian target of rapamycin) pathway activation and then inhibition of caspase-
3 [126]. Podosome assembly, vesicle transport, secretion of proteases, and organisation of
microtubules are all regulated by c-Src pathway activity [127]. For osteosarcoma, inhibition of c-
Src induces apoptosis and inhibits invasion in vitro. Primary tumour volume in a murine model
of osteosarcoma was also reduced by c-Src inhibition [128].

Osteoclast pathways of differentiation, maturation, and activation have potential as therapeutic

targets. Inhibition of bone resorption at the tumour-bone interface may lead to reduced local
invasion by osteosarcoma. The central role that RANKL plays in osteoclast function makes it a
particularly attractive target. Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and
strongly suppresses osteoclast differentiation both in vitro and in vivo [129]. OPG gene therapy
has been applied to a murine model of osteosarcoma and successfully suppressed osteolytic
activity. There were a reduced number of osteoclasts associated with tumours, leading to reduced
local osteosarcoma progression and improved survival