COMPLEX CASE ANALYSIS: Clear Cell Sarcoma By Anastasia Lezhanska

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Complex Case Analysis: Clear Cell Sarcoma

By: Anastasia Lezhanska 

 

Clear Cell Sarcoma, EWSR1- CREB1 Fusion, and CDKN1A

 

Clear cell sarcoma (CCS) is a malignant, soft tissue tumor, with approximately half of patients subsequently developing lung or lymph node metastases. The most distinctive genetic alteration of CCS is the fusion of ATF1 and EWS genes due to chromosomal translocation.

EWSR1 is a housekeeping gene and the encoded protein of this gene is mainly found in the nucleus of the cell. While its role is not fully known, it is involved in various processes and interacts with TFIID subunits, RNA polymerase II, parts of the spliceosome, mitotic spindles, and microtubule stabilization. It is also involved in meiosis with proposed functions in DNA pairing, recombination and repair mechanisms. Additionally, it has a role in cellular senescence and so is a vital gene that aids in maintaining proper cell growth and aging.

When fusion products occur between EWSR1 and different partner genes, usually the RNA-binding domain of EWSR1 is detached and substituted with the DNA-binding domain of the fusion gene partner4. The variant translocation involving EWSR1 and CREB1 genes appears to be associated with sarcomas located in the GI tract. Both the ATF1 and cAMP-responsive element-binding protein (CREB) are part of the leucine zipper superfamily of transcription factors. Overexpression of CREB is associated with the acquisition of metastatic potential by melanoma cells, which may contribute to the similarities between clear cell sarcoma and malignant melanoma. It is thought that the products of these gene fusions are novel, tumor-specific chimeric transcription factors that can lead to deregulated gene expression5.

Upregulation or gain of function mutations in genes that cause repression of CDKN1A transcription may also contribute to cancer development. Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a tumor suppressor gene and loss of its function results in the inactivation of p53 pathways which leads to uncontrolled cell proliferation9. Currently, there is no standard treatment option for tumors with CDKN2A alterations.

 

p21 Inhibitors

 

p21 is an important cyclin-dependent kinase inhibitor for the G1-S transition of the cell cycle. It can stop the cell in G1 phase to allow it to repair damaged DNA before continuing to S phase. In one study, its activity increased significantly after treating cells with the EWS-Fli1 fusion gene, another variant of Ewing’s sarcoma. p21 can induce differentiation of cells and can supress the growth of malignant cells in vitro and in vivo7. The expression of p21 is positively regulated by the tumor suppressor gene product p53.

Recent studies also suggest that p21 can act as an oncogene under certain conditions and cytoplasmic p21 exhibits anti-apoptotic activity by inhibiting proteins involved in apoptosis8. p21 mediates biological activity mainly by binding to and inhibiting the kinase activity of the cyclin-dependent kinases (CDKs). Moreover, by binding to proliferating cell nuclear antigen (PCNA) it interferes with PCNA-dependent polymerase activity, thus inhibiting DNA replication and regulating different PCNA-dependent DNA repair processes.

Several anticancer drugs, such as histone deacetylase (HDAC) inhibitors function through their ability to promote induction of p21. Other such agents include statins which are regularly used to lower cholesterol levels and display substantial anti-proliferative capability by inducing p21. These are currently being studied for their anti-tumorigenic functions. However, it is challenging to find agents that will induce only the tumor-suppressor functions of p21 and not its oncogenic activities. A possible solution to this dilemma is to target factors upstream or downstream of p21 pathways which also affect aspects of p21 function. Alternately, it may be effective to utilize the ability of p21 to cause senescence in tumors, for example by re-establishing p53 tumor-suppressor function.

 

Nanthealth

 

            Nanthealth is an organization working with Providence Health System which is an initiative focusing on whole genomic sequencing. Together, they aim to provide patients and healthcare providers with the most complete information on the genomic and proteomic characterization of the patients and their cancer tissue. Nanthealth utilizes novel technology that can analyze tumors at the molecular level and makes recommendations for treatments that are most likely to successfully target the patient’s specific cancer. The results of their analyses are assimilated using algorithms that are capable of recognizing disrupted pathways particular to that tumor. Disrupted pathways, prognostic markers, and possible treatments are all presented to the oncologist in a report that is easy to understand using Nanthealth’s eviti2. Eviti is an application that provides the healthcare team and the patient with information on all possible treatment plans and costs associated with their specific condition so that they can make better decisions about their healthcare plan.

 

Champions Oncology

 

Champions Oncology is a novel technique used to test various cancer treatments on mice and it allows for testing of a model that is more reflective of the true nature of a patient’s cancer. It works by implanting a tumor graft, taken from a patient during a biopsy or surgery, under the skin of an immunodeficient mouse. While the mouse is given time to get accustomed to the tumor graft and grow, the patient may be recovering from surgery and undergoing treatment as suggested by their healthcare team. After the mouse is ready for testing, Champions Oncology exposes it to various potential treatments and assesses the response of the tumor within the animal. This technique can provide valuable information about which treatments may potentially work on the patient and can help eliminate therapies that do not appear to fight the tumor.

There are some drawbacks to using this technique, however, including the fact that the tumor grafts are inserted under the skin of mice and not in specific organs where the cancer would normally be found in a human patient. Therefore, these mice do not accurately mimic the environment of the human tumor which may affect how the tumor responds to the treatments that are tested. Furthermore, the mice have highly impaired immune systems in order for them to tolerate the human tumors. However, this means that they cannot reflect how a human immune system would respond to a treatment and cannot be used to test immunotherapies.

 

Drugs/Combination of Drugs

 

            The most effective drugs for soft tissue sarcomas include doxorubicin which, in combination with other drugs such as ifosfamide, demonstrates an increased overall response in the majority of patients, although the effect on overall survival is unknown11. These drugs are the standard in treating soft tissue sarcomas but there are other options.

The most promising treatments for CCS are targeted therapies which aim to fight features of CCS cancer cells specifically. One type of targeted therapy is a receptor tyrosine kinase inhibitor which inhibits overactive signalling molecules in cancer cells that promote uncontrolled growth. An example of this is crizotinib, which is currently being tested in clinical trials, and pazopanib – a drug that was tested in an experiment utilizing a novel CCS cell line called Hewga-CCS1. Hewga-CCS was developed from skin metastatic lesions of a CCS female patient and xenografts were established and characterized by immunohistochemical reactivity. The experiment conducted on the Hewga-CCS line provided important information on the antitumor effects of pazopanib.

Another type of targeted therapy is epigenetic therapy which targets enzymes that chemically modify DNA such as the histone deacetylase inhibitors mentioned earlier. Other drugs include dacarbazine, an alkylating agent which works by adding an alkyl group to the DNA of cancer cells, thereby destroying them. It may also inhibit DNA synthesis by acting as a purine analog, however it is not very effective on its own and is better used in combination with other drugs. Gemcitabine is a pyrimidine antimetabolite and has a favourable toxicity profile. It inhibits processes required for DNA synthesis by getting incorporated into a growing DNA chain and then allowing one additional nucleotide to be incorporated before DNA polymerases stop and are unable to proceed12. This is known as masked termination and is an ingenious mechanism of action because the DNA polymerases cannot recognize gemcitabine as the problem since it is masked by the final nucleotide incorporated after it in the DNA chain.

 

Side-Effect-Free Chemo

 

Dr. Matsumura of Berkeley Institute International developed Side-Effect-Free Chemo – a chemotherapy that simultaneously aims to treat cancer and eliminate the toxic effects of standard chemotherapy. It works by protecting normally dividing cells in the patient’s body with an antidote from the chemotherapy while it targets cancerous tissue. One of the most significant side-effects of traditional chemo is bone marrow suppression, which also prevents the use of chemo in high doses. However, Side-Effect-Free Chemo would prevent the loss of normal immune cells and would enable the neutrophil cascade to occur, thereby helping the patient’s immune system to combat the cancer. The neutrophil cascade is an innate immune response where circulating neutrophils detect pathogens and mark them for destruction by other neutrophils. Side-Effect-Free Neutrophil-Potentiated Chemotherapy works by protecting neutrophils from the toxicity of chemotherapy and may kill up to 95% of cancer cells compared to only 60% killed by standard chemotherapy13.

 

 

 

 

 

 

 

 

 

References

 

  1. Outani H., Tanaka T., Wakamatsu T.,  Imura Y., Hamada K., et al. Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS. BMC Cancer. 2014, 14: 455.
  2. Nanthealth, LLC. 2015. URL: nanthealth.com
  3. Champions Oncology Inc. 2015. URL: championsoncology.com
  4. Romeo S., Dei Tos AP. Soft tissue tumors associated with EWSR1 translocation. Virchows Arch. 2010, 256: 219-234.
  5. Thway H., Fisher C. Tumors with EWSR1-CREB1 and EWSR1-ATF1 fushions: the current status. Am J Surg Pathol. 2012, 36: 1-11.
  6. Thway K., Nicholson AG., Lawson K., Gonzalez D., Rice A., et al. Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion: a new tumor entity. Am J Surg Pathol. 2011, 35: 1722-1732.
  7. Nakatani F., Tanaka K., Sakimura R., Matsumoto Y., Matsunobu T., et al. Identification of p21WAF1/CIP1  as a direct target of EWS-Fli1 oncogenic fusion protein. J Biol Chem. 2003, 278(17): 15105-15115.
  8. Abbas T., Dutta A. p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer. 2009, 9(6): 400-414.
  9. Personalized Cancer Therapy: Knowledge Base for Precision Oncology. The University of Texas MD Anderson Center. 2015. URL: pct.mdanderson.org/genes/cdkn2a/show
  10. Barry G., Neilsen TO. Learn about sarcoma. The Liddy Shriver Sarcoma Initiative. URL: sarcomahelp.org/clear-cell-sarcoma.html
  11. Eriksson M. Histology-driven chemotherapy of soft-tissue sarcoma. Ann Oncol. 2010, 21(7): 270-276.
  12. Plunkett W., Huang P., Heinemann V., Grunewald R., Gandhi V. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol. 1995, 22: 3-10.
  13. Side-Effect-Free Chemotherapy. Berkeley Institute International. 2014. URL: http://www.cancer-institute.com/index.php

Chemotherapy Side Effects. MasotheliomaGuide. 2015. URL: http://www.mesotheliomaguide.com/treatment/chemotherapy