Mitchell+Dang

** Parosteal Osteosarcoma **
Augustus was excited to begin summer training for his final season as the quarterback for his football team. Practice was going as he wanted it to and he finished at the top of his team with the best 40-yard dash time and the highest bench press. He was expecting to play the best season of his life until things took a turn. About three weeks into summer training, Augustus began feeling some pain in his right leg and thought it was just from a fall he had the day before. He followed his trainer’s instructions by icing his leg and avoided putting pressure on it for a few days. After a few days, the pain persisted and became more severe, to the point where he could barely walk. Worried that he may not be able to play his final season, Augustus went to see his doctor. After taking x-rays, his doctor noticed a cauliflower-like mass in a region on his posterior distal femur. Not wanting to jump to any conclusions as this mass can very well just be an infection, Augustus’ doctor recommended him to an orthopedic surgeon at Stanford University. Augustus underwent an MRI scan so his doctor could get a better image of the cauliflower mass on his femur and confirm what the mass could be. What Augustus and his parents thought was just an injury from practice turned into their worst nightmare. Augustus was diagnosed with parosteal osteosarcoma (POS) on August 3, 2015, just two weeks before the start of his final season.
 * __The Cancer__ **

As any patient and their family would do after hearing this news, questions after questions were thrown at the doctors working with Augustus. His face was blank listening to his doctors explain to him this disease. Osteosarcoma is a cancer of the bone that mostly affects children and young adults, but can affect people of any age. In the United States, there are around 800 new diagnoses of osteosarcoma each year and half of those cases are children and teenagers. [1] The disease usually starts in osteoblasts, which are bone cells that become new bone tissue, and typically develops where the bone is growing quickly. Osteosarcoma has different subtypes that can be identified through magnetic resonance imaging and biopsies. MRIs give the doctors a more detailed image that helps them determine if there are any possible metastases. There are three subtypes that differ in how fast the cancer grows: high-grade osteosarcomas, intermediate-grade osteosarcomas, and low-grade osteosarcomas. [2] Parosteal osteosarcoma is classified as a low-grade, surface osteosarcoma and arises from the periosteum, which is the membrane that covers the outer-surface of bones. It is a rare form of osteosarcoma but is the most common among surface osteosarcomas. [3] Periosteal osteosarcoma is another surface osteosarcoma that has a higher grade than parosteal osteosarcoma. Pain and swelling is worse in this variant and “runs a rapid course when compared to parosteal osteosarcoma.” [3] While all types of osteosarcoma require surgical resection, parosteal osteosarcoma normally does not need to be treated with chemotherapy if the cancer is diagnosed in its early stages. Overwhelmed with all this information, Augustus sat across from the doctors and asked why this happened to him. How could a healthy, athletic, and seemingly invincible star quarterback get tackled by such a disease?

Researchers have not found a clear cause for osteosarcoma but some are hypothesizing that mutations that turn off the TP53 tumor suppressor gene gives people a higher risk of developing certain cancers including osteosarcoma. [4] Although there is no clear cause, there are several risk factors that may have contributed to Augustus’ osteosarcoma. Genetic predisposition may be one risk factor but seems very unlikely because none of his family members have developed this disease. His doctors believe that his age and gender may have possibly been a key factor in his diagnosis as the risk for osteosarcoma is highest in teenagers who are right in the middle of their growth spurts. From checking his past physical exam records, his doctors noticed a growth in height between the ages of 16 to 17. His mountainous height of six feet and four inches is perfect for being the star quarterback but also perfect for osteosarcoma to develop which suggests that rapid bone growth is related to osteosarcoma. [5] One study determined that height played a significant role in osteosarcoma by collecting data from fourteen published journals that included the height of patients at diagnosis. It was found that being taller than average (51th-90th percentile) and very tall (>90th percentile) played a factor in patients with osteosarcoma. [23]

At this point in their meeting, Augustus and his parents were exhausted. It seemed that no good news would come out of this. However, the doctors provided information that changed the entire mood of the room. They explained that parosteal osteosarcoma is a low-grade subtype meaning Augustus’ prognosis is bright. With an 80-95% five year survival rate, his doctors are certain that Augustus will be able to move past this part of his life. His treatment will require only an “upfront wide excision without adjuvant chemotherapy [since he has a low grade tumor].” [6] After the surgery, he would need to have routine checkups in order to ensure that there are no more cancerous cells in his leg as well as physical therapy to ensure normal function of his leg. This bright news certainly brought a glimmer of hope back into Augustus’ eyes. Although he would be missing his final season due to the surgery, he is glad that he already has the upper-leg in this fight.

__** Molecular Basis **__ After coming home, Augustus decided to research further about parosteal osteosarcoma. He discovered that mutations in the tumor suppressor gene, TP53, play a significant role in the formation of osteosarcoma. The protein that TP53 codes for, p53, causes the cell to “arrest the cell cycle [when it senses DNA damage] and either repair the damage or induce a series of events that lead to cellular death or senescence,” essentially acting as a checkpoint for the cell cycle [7].

The amplification of MDM2 can also be seen in patients with parosteal osteosarcoma. Located on chromosome 12q15, this gene codes for a nuclear-localized ubiquitin ligase which targets tumor suppressor proteins and degrades them. The loss of tumor suppressor proteins will contribute to the rapid and uncontrolled proliferation of cells. The oncoprotein MDM2binds to p53 and promotes its degradation as well as downregulation in transcription [8]. In a growing teenager like Augustus, whose cells are constantly and rapidly dividing, it is important that p53 is functioning properly to ensure that no damaged DNA is passed on to dividing cells and that tumor development can be suppressed. For the inhibition of p53 to continue, there must be a mechanism to produce a high number of MDM2 proteins.



In another study, 100% of the parosteal osteosarcoma (POS) patients showed gain of chromosome 12 sequences where the MDM2 gene is located. Five of the six patients had a supernumerary ring chromosome with an “increase of DNA sequence copy number confined to chromosome 12 […] indicating that gains and high level amplifications seen at chromosome 12 in POS are most likely related to the presence of supernumerary ring chromosomes” [9]. Ring chromosome formation is thought to be a result of telomere shortening and breakage-fusion-bridge (BFB) events which consequently can cause cancerous mutations to arise. When telomeres begin to shorten, the ends of the chromosome may become reactive and trigger a fusion event that forms a ring. Ring formation may also form through breaks in the arms of the chromosome and the fusion of those broken ends (Figure 2). During mitotic division, a ring chromosome will form a bridge during anaphase and follow either of these two paths that lead to genomic instability: the ring breaks and fuses with the ends to form yet another ring or the ring undergoes nondisjunction in which the size of the ring will double [10]. These rings continue through more BFB events which leads to potential gene amplification and explains the cause of high amplification levels of the MDM2 gene seen in the previously mentioned study. Figure 1 reveals that MDM2 is highly amplified and that there is a difference in sizes due to nondisjunction. Different hallmarks of cancer affect each other in like the gears of a machine. This positive feedback loop starts with telomere shortening which leads to the formation of rings and the amplification of MDM2. As the ring chromosomes go through mitosis, the number of MDM2 gene increases and there is sufficient inhibition of p53. This allows breakage events and nondisjunction to occur without cell cycle arrest or apoptosis, ultimately leading to the development of parosteal osteosarcoma.

MDM2’s location on chromosome 12q15 means that it is much closer to the centrosome than the telomeres. This contradicts Szymanska et. al’s hypothesis that MDM2 amplification is caused by telomere shortening since telomeres are located at the ends of chromosomes. Double-strand breaks give a better explanation for MDM2 amplification instead. In Figure 2a, these breaks can happen anywhere and then fusion of these broken ends occur and cause the gene to be amplified. Because p53 is inhibited due to the overamplification of MDM2, the cell is not able to repair this damaged DNA or induce apoptosis of the cell and this DNA is able to be passed on to more generations.



Another hallmark of osteosarcoma is its ability to evade growth suppressors. Cyclin dependent kinases (CKDs), cyclins, and pRb play a role in the cell cycle by acting as a checkpoint of the cell cycle through mainly external factors such as growth factor signals. CDK4 must bind with its respective cyclin to become active, which in turn phosphorylates pRb, allowing the cell cycle to progress by releasing the E2F growth factor. In POS, RB1 is inactivated in at least half of the tumors through mutations, meaning there is no checkpoint in the cell cycle. While the CDK and cyclin unit promote proliferation, RB1 and p16 suppress cell proliferation. P16 is the product of INK4A and acts as the inhibitor of CDK4 in order to downregulate CDK function.In many studies, it is shown that p16 “is inactivated in osteosarcomas that lack RB mutations”and that chromosome 9p21 undergoes deletion in 20% of osteosarcomas [11]. This very regulated p16-pRB-cell cycle pathway becomes deregulated in osteosarcomas and the lack of either of these growth suppressors leads to tumor formation.

A much more recent study has shown that mutations in the GNAS gene may play a role in parosteal osteosarcoma. In 9 cases of parosteal osteosarcoma, direct sequencing revealed GNAS activating mutations in 5 cases [12]. “GNAS activating mutations are missense mutations that lead to amino acid substitution of either residue Arg 201 or Gln 227 ” which are located on the alpha subunit of the G protein and affect the “turn off” mechanism [13]. GNAS helps produce the alpha subunit of the guanine nucleotide binding protein (G protein) which triggers many signaling pathways that “ultimately influence many cell functions by regulating the activity of hormones.” [14] The alpha subunit is important in stimulating the enzyme adenylate cyclase which plays a major role in pathways that regulate the development of bones. Essentially, this enzyme prevents the production of bone tissue in the wrong areas. The GNAS activating mutation may be the reason for Augustus’ tumor formation on the periosteum (outer membrane of bone) of his distal femur.

Augustus sat in his chair trying to process all this information he had just learned. Although his doctors said that his prognosis is bright and his treatment would only require surgical resection, he wonders what would have happened if he had been diagnosed later and the cancer was able to dedifferentiate and become high grade. Could these mutations have transformed his cancer into something worse if he had not gone to the doctor sooner? He felt overwhelmed with all these negative thoughts and scenarios playing through his head and tried to pull away from the cancer’s grasp on his mind.

__** Treatment **__ Regardless of its grade, parosteal osteosarcoma requires surgical resection for treatment. Normally there is no need for adjuvant chemotherapy. Limb-sparing surgery and amputation are essential treatment for osteosarcoma patients and is most recommended by oncology orthopedic surgeons. Studies have shown that limb-sparing surgery results in more favorable outcomes while amputations are usually performed for patients who have tumors in “unfavorable anatomical locations.” [12] In limb-sparing surgery, the tumor in the bone is removed and is replaced by either an allograft bone, a prosthesis, or both. These metallic devices are fit in with the rest of the bone using bone cement or allowing the bone to grow into the new part. [13] Because Augustus has a low-grade lesion, he will luckily only have to undergo limb-sparing surgery. However, his leg will most likely not return to the same level of function after surgery- shattering his hopes of playing football again. He will have to have regular check-ups for many years which will include x-rays, blood tests, and scans to be sure that the cancer has not metastasized.

In many cases other than Augustus’, patients have had to undergo some sort of neoadjuvant or adjuvant chemotherapy following their surgery which improved the outcomes of those who had localized osteosarcoma. A randomized control trial conducted by the European Osteosarcoma Group showed that the combination of doxorubicin and cisplatin had better tolerance in patients than another multidrug regimen with no difference in three year and five year overall survival rate. [12] Doxorubicin is an anthracycline antibiotic that enters the nucleus of the cancer cell and poisons the topoisomerase-II which leads to DNA damage and apoptosis. [14] Topoisomerase-II’s function is to unwind the supercoils in DNA for transcription so when it is poisoned by doxorubicin, DNA is unable to reseal which stops transcription. [15] One of the most important side effects of doxorubicin is its cardiotoxicity to the patient. Doxorubicin’s cardiotoxicity causes the heart to be less efficient at pumping blood, which ultimately limits the therapeutic potential of the drug. [16] Cisplatin interacts with DNA and forms DNA adducts, which are DNA segments ionically bonded to a carcinogen, and activates the p53 signal transduction pathway. [17] When cisplatin binds to the DNA, it causes crosslinking and ultimately leads to apoptosis if this damage is not repaired. As with any chemotherapy drug, cisplatin affects normal cells and most importantly reduces the number of blood cells leading patients to become more prone to infection. [18] Overall, the combination of doxorubicin and cisplatin seem to be a good option for osteosarcoma patients as many studies have shown that it is better tolerated by the patient. However, due to the cardiotoxicity of doxorubicin, patients should attempt to find an alternative.

Another treatment for parosteal osteosarcoma would be to target MDM2 using small molecule inhibitors such as Nutlin-3 with cisplatin. Nutlin-3 is a non-genotoxic antagonist of the MDM2-p53 interaction which has been used in many preclinical studies. This compound “targets a small hydrophobic pocket on MDM2” where p53 would normally bind to. [19] This leads to p53 downstream transcriptional targets which play a role in apoptosis and cell cycle arrest. It is often better for these MDM2 antagonists to be combined with other standard chemotherapy drugs that target the growth of mutated TP53 cells. The antagonists alone have the potential of causing the cancer to develop resistance to it. It has been suggested that resistance is acquired through the “prolonged exposure of cells to sub-lethal doses through de novo inactivating TP53 mutations.” [20] Combined therapy has many benefits such as dose reduction, toxicity reduction, and synergistic therapeutic effects. This is why one of these antagonists combined with cisplatin could be a better alternative than doxorubicin and cisplatin. Although still in preclinical phases, early research shows that nutlin-3 protects kidney cells from apoptosis caused by cisplatin. [21] This finding shows that nutlin-3 can improve the therapeutic efficacy in combined therapies by reducing the side effects caused by standard chemotherapy drugs. By preventing apoptosis in normal cells, nutlin-3 gives a promising future for cancer patients undergoing chemotherapies that have severe side effects.

One study has shown that nutlin-3 alone is effective in restoring the p53 pathway in cells with wild-type p53. Rather than inducing p53 at the mRNA level, p53 was increased at the protein level. [22] These results are consistent with the previously mentioned finding that nutlin-3 targets an area on MDM2 that interferes with the MDM2-p53 interaction and prevents p53 degradation. Nutlin-3 seems to have great potential as a drug that can treat osteosarcoma and many other cancers but requires more studies to ensure its efficacy. Nutlin-3 alone most likely will not be as effective as nutlin-3 combined with other chemotherapy drugs because targeting one single pathway can’t destroy a whole cancer. Just as cancer is a multi-step process, cancer treatment is a team effort that requires many different weapons to kill the many-headed beast. Two years after his surgery, Augustus is completely free of any cancerous cells. He has been returning to the doctor for routine checkups to ensure that his cancer has not returned or spread. He regained full function of his leg again after months of physical therapy and returned to football shape. The process was not easy but it was not the hardest task he has ever had to do. The cancer made him realize that no matter how healthy, active, and young you are, cancer can strike any time. When that moment comes, you just have to be hopeful and prepared for what lies ahead. __** Apercu **__ Every cancer is different and unique to the patient; some can be prevented while others cannot. Unfortunately, Augustus' parosteal osteosarcoma could not have been prevented but he was lucky enough to have it spotted at early stages and have the tumor completely removed. The most accurate diagnosis and best treatment leads to a more controllable disease that can possibly be cured.

__ **References** __ >
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