Chromosomal Translocation and Ewing’s Sarcoma, Essay Example

Ewing’s sarcoma is a bone tumor that typically occurs in pediatric patients. Unlike many other bone cancers, 85% of Ewing’s sarcoma cases exhibit a precise chromosomal translocation that result in this malignancy. Specifically, tumorigenesis is the consequence of translocation between chromosomes 11 and 22, which fuses the EWS gene of chromosome 22 to the FLI1 gene of chromosome 11. Researchers continue to evaluate this translocation in an attempt to determine what drives the formation of this oncogene and to determine ways that drugs can prevent formation of the aberrant protein during translation.

Ewing’s sarcoma is considered a rare cancer and disease survival rate has increased from 59% to 76% for children younger than 15 years and from 20% to 49% for adolescents aged 15 to 19 years since the 1970’s (Smith et al., 2010). Despite the rarity of the disease, researchers are concerned with developing effective treatments because of the high mortality rates associated with it. Prior to the 1970’s, pediatric oncologists attempted to cure this tumor using surgery and single agent chemotherapy. However, after considering the results of many research protocols initiated in this decade, it was found that multi-agent chemotherapy was a more useful technique. While this helped improve the five-year survival rate of these patients significantly, there are many patient subgroups who fail to demonstrate improvement. Therefore, there is a need to develop new treatment modalities that compensate for this difference in survival.

While chemotherapy methods typically target tumors based on their ability to grow more quickly than normal cells, these treatments typically cause many side effects and may allow the tumor to grow resistant over a long period time after repeated treatment. The use of multi-agent chemotherapy makes drug resistance take longer, but ultimately, these drugs stop benefiting the patient. Therefore, many researchers are calling for a need to develop methods that target the patient’s individual tumor therapy. To do so, many studies aim to identify biomarkers that can be specifically targeted to confer therapeutic benefit. Since the chromosome 11-22 translocation is common in a majority of tumors, it would be useful to develop therapy that targets this abnormality and to combine this treatment with standard therapy.

Several clinical tests are used by physicians to diagnose Ewing’s sarcoma. However, it is essential to note that this tumor is not typically detected in early stages due to the fact that symptoms are mild. Symptoms typically include pain and swelling and do not cause the patient any alarm or lead them to believe they should visit a physician. Therefore, by the time this cancer is diagnosed, it has likely already spread to other tissues. The first test that is used includes x-rays, magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, and bone scans. This will allow the physician to determine whether parts of the bone have been destroyed. Next a biopsy will be performed to determine the presence of cancer cell histology. Lastly, blood tests, CT scans of the lungs, bone scans, and a bone marrow biopsy may be performed to determine whether the tumor has spread.

Physicians do not conventionally test patients who are expected to have Ewing’s sarcoma for the presence of the genetic translocation. However, researchers often aim to collect this data to verify disease status and to determine how this translocation could be targeted for treatment. Therefore, this information is typically only available for patients who have been enrolled on a research protocol and agreed to genetic testing. In these studies, a variety of laboratory methods are used to assess the status of the 11-22 genetic translocation. These include integrated clinicopathologic, cytogenetic, fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR) techniques (Warren et al., 2013).

The precise base pair location in which the translocation occurs in Ewing’s sarcoma varies from patient to patient. However, since it occurs between the EWS gene of chromosome 22 to the FLI1 gene of chromosome 11, the sequences of these two genes are essential in determining whether the translocation is present in patients. The amino acid sequence for the EWS-FLI1 translocation is as follows: PTSYPPQTGSYSQAPSQYSQQSSSYGQQNPYQILGPTSSRLANPGSGQIQLWQFLLELLS DSANASCITWEGTNGE

The nucleotide sequence for the EWS-FLI1 translocation in one patient with Ewing’s sarcoma is as follows:

CCCACTAGTTACCCACCCCAAACTGGATCCTACAGCCAAGCTCCAAGTCAATATAGCCAACAGAGCAGCAGCTACGGGCAGCAGAATCCGTATCAGATCCTGGGCCCGACCAGCAGTCGCCTAGCCAACCCTGGAAGCGGGCAGATCCAGCTGTGGCAATTCCTCCTGGAGCTGCTCTCCGACAGCGCCAACGCCAGCTGTATCACCTGGGAGGGGACCAACGGGGAGT

In order to gain more information about this genetic translocation, it would be useful to determine the similarity between the nucleotide sequences of a large number of patients. To do so simply, restriction digest experiments can be employed. Each patient tumor sample will be labelled numerically in a tube and without patient identifiers. Patients without a formal cancer diagnosis should provide blood or saliva for testing. The Qiagen kit and protocol for DNA extraction from tissue samples will be utilized. PCR will be used to generate copies of the EWS-FLI1 genetic translocation using CAGGTGATACAGCTGGCGTT as the forward primer and GGTCCCCTCCCAGGTGATAC as the reverse primer. The BigDye Direct Cycle Sequencing kit will be used to prepare this translocation for sequencing, which will verify the presence of the gene. If the genetic translocation observed appears close to what is expected, it will be included in the analysis.

In the first experiment, all patient samples will be analyzed via restriction digestion using SpeI and AvaII. These enzymes will be incubated with the patient DNA for approximately an hour at 37 degrees Celsius. They will then be put on ice and a gel for analysis will be prepared using 5% agarose. The negative control will be a section of DNA without sites for either of these two enzymes to ensure that there is no cutting when these sites are not present and the positive control will be a section of DNA with sites for both of these enzymes to ensure that proper cutting is taking place. This digest will result in a band of approximately 200 and one very small band of 19 for the patient sample whose sequence was displayed above. It is possible that this band will vary for other patients, which will indicate that the genetic translocation is different for different individuals.

The second experiment will be a repeat of the first using different restriction enzymes. This time, only BanII will be used which will result in two bands of approximately 100 nucleotides for the example shown above. Results that vary from this band size compared to controls with and without BanII sites will demonstrate that the EWS-FLI1 translocation sequences vary from patient to patient. These tests will help define the parameters that Ewing’s sarcoma patients with the EWS-FLI1 translocation exhibit, which will give physicians a quick and meaningful way to test for the presence of the disease.

Works Cited

Smith MA, Seibel NL, Altekruse SF, et al. “Outcomes for children and adolescents with cancer: challenges for the twenty-first century”. J Clin Oncol 28.15 (2010): 2625-34. Print.

Warren M1, Weindel M, Ringrose J, Venable C, Reyes A, Terashima K, Rao P, Chintagumpala M, Hicks MJ, Lopez-Terrada D, Lu XY. “Integrated multimodal genetic testing of Ewing sarcoma–a single-institution experience”. Human Pathology 44.10 (2013): 2010-9. Print.