Cytarabine for the Treatment of Ewing's Sarcoma

Steven G. DuBois, MD

Holcombe E. Grier, MD

Kimberly Stegmaier, MD

Division of Pediatric Hematology/Oncology

Dana-Farber Cancer Institute

Children’s Hospital, Boston

Introduction

Ewing's sarcoma is the second most common bone cancer in children and young adults, after osteosarcoma. A biologically equivalent tumor, known as primitive neuroectodermal tumor (PNET), arises in the soft tissues. Patients with Ewing's sarcoma and PNET require systemic therapy with chemotherapy and local therapy with surgery and/or radiation therapy. In recent years, the outcome for patients without metastases at initial diagnosis has improved considerably. Currently, approximately 70% of these patients can be expected to survive without disease recurrence. Even with intensive treatment, though, up to 30% of patients with localized Ewing's sarcoma ultimately have tumors that do not respond to therapy or that recur after finishing treatment.¹ Approximately 25% of patients have metastatic disease at diagnosis and the outcome is much worse for these patients. Up to 80% of patients with initially metastatic disease have refractory or recurrent tumors.²

Patients with suspected Ewing's sarcoma typically undergo imaging of the primary lesion followed by biopsy of the primary lesion. In the absence of metastatic disease, newly diagnosed patients begin treatment with neoadjuvant combination chemotherapy. In North America, the current standard chemotherapy consists of cycles of vincristine, doxorubicin, and cyclophosphamide alternating every 3 weeks with cycles of ifosfamide and etoposide. After approximately 12 weeks of chemotherapy, patients undergo local therapy to the primary lesion. Local control may take the form of surgical resection, definitive radiation therapy, or a combination of surgery with radiation. Following local control, patients continue to receive chemotherapy until they have received a total of 14 cycles of therapy.

Sidebars that appear in boxes with red borders are directed towards physicians and nurses. However, patients and caregivers may find some of them informative.

Ewing's sarcoma and soft tissue PNET are characterized by unique genetic changes in tumor cells that are thought to be crucial for the development of these cancers. The majority of these tumors harbor a genetic abnormality known as a translocation.

The article "Chromosomal Translocations in Sarcomas: New Perspectives" by Dr. Mercado and Dr. Barr that appeared in the February 2006 issue of ESUN provides a nice introduction to translocations.

Sidebars that appear in boxes with blue borders are directed towards patients and caregivers. However, physicians and nurses may find some of them useful as well.

The most common translocation seen in Ewing's sarcoma fuses parts of two genes together, the EWSR1 gene to the FLI1 gene.³ In a smaller number of tumors, the EWSR1 gene fuses to other genes similar to the FLI1 gene. These translocations involving the EWSR1 gene result in the production of proteins known as EWS fusion oncoproteins. EWS fusion oncoproteins likely drive the development of Ewing's sarcoma and soft tissue PNET. Targeting these EWS fusion oncoproteins for the treatment of Ewing's sarcoma would theoretically be an attractive approach to this disease. Currently, the effects of these oncoproteins in tumor cells are not clear, making targeted drug development for this disease difficult. A new way of discovering effective drugs in cancer has identified a drug known as cytarabine as a possible targeted therapy for Ewing's sarcoma.

Cytarabine, also known as cytosine arabinoside or ara-C, is a chemotherapy drug that belongs to a family of drugs known as nucleoside analogues. Once inside a cell, cytarabine undergoes changes to its active form known as ara-CTP.⁴ Once incorporated into DNA, ara-CTP blocks DNA production directly by impeding the growth of the DNA chain. Cytarabine is used at a wide range of intravenous doses, with current regimens ranging from 100 mg/m²/dose to 3 grams/m²/dose.⁴ Common side effects observed with cytarabine include decreased blood counts, hair loss, nausea, vomiting, mucositis, rash, and elevation in liver enzymes. With high dose cytarabine, patients may experience cerebellar toxicity and irritation of the eyes.^5-8

Cytarabine has typically been used in the treatment of leukemia and lymphoma. More recently, laboratory research suggests that cytarabine may be useful for patients with Ewing's sarcoma. In this article, we review the preclinical evidence indicating a possible role for cytarabine in the treatment of Ewing's sarcoma. We also discuss the limited clinical experience with this drug in patients with Ewing's sarcoma. Finally, we provide an overview of a current Children’s Oncology Group (COG) phase II study prospectively evaluating cytarabine in patients with Ewing's sarcoma.

Preclinical Evidence Demonstrating Efficacy of Cytarabine in Ewing's Sarcoma

Laboratory experiments suggest that cytarabine may be effective in treating patients with Ewing's sarcoma. Hofbauer and colleagues evaluated the sensitivity of Ewing's sarcoma and PNET cell lines to a range of chemotherapy drugs.⁹ Cells were treated with various drugs for 48 hours and cell growth was measured. Cytarabine at very low doses effectively decreased the growth of Ewing's sarcoma and PNET cells. In order to confirm their findings, this group also examined cells under the microscope and demonstrated that the cells died in response to cytarabine.⁹

More recently, Stegmaier and colleagues have developed a method of identifying effective chemotherapy drugs using gene expression profiles.¹⁰

The article "Gene Profiling: Unlocking the Inner Workings of Sarcoma Cells" by Dr. Mackall and Dr. Kahn that appeared in the October 2004 issue of ESUN provides a nice introduction to gene profiling.

Using this technique, the group evaluated drugs that might affect the EWS/FLI fusion oncoprotein. Specifically, a laboratory method known as RNA interference was first used to block the production of the EWS/FLI fusion oncoprotein in Ewing's sarcoma cells.

The 2006 Nobel Prize in Physiology or Medicine was awarded to Professor Andrew Z. Fire at Stanford University, California, USA, and Professor Craig C. Mello at the University of Massachusetts Medical School in Worcester, USA. They receive the prize for their discovery that double-stranded RNA triggers suppression of gene activity in a homology-dependent manner, a process named RNA interference (RNAi). Click here to read an interesting account of this discovery. An animated tour of the process of RNA interference is available on the Nature Publishing Company's website (click here; note that you can either view the animation online or download it). The NOVA website also contains an interesting and instructive video on RNAi A brief, but interesting history of RNA interference, "Cancer Concepts: RNAi", is on the American Association for Cancer Research's website.

In response to EWS/FLI fusion oncoprotein blockade, cells either died or showed decreased cell growth (Figure 1).

Figure 1: Schematic diagram of RNA interference. Ewing's sarcoma cells expressing the EWS/FLI oncoprotein were grown in culture. These EWS/FLI “active” cells were treated with interfering RNA to block the expression of EWS/FLI. Surviving cells were maintained in culture as EWS/FLI “inactive” cells.

For those cells that survived, an EWS/FLI “inactive” gene expression profile was generated using DNA microarrays (Figure 2).

Figure 2: Schematic diagram of gene expression profiles. The gene expression profile of EWS/FLI “active” cells differs from the gene expression profile of EWS/FLI “inactive” cells.

Next, a collection of chemicals was screened to determine if any could produce this same EWS/FLI “inactive” profile in Ewing's sarcoma cells. Cytarabine was one of the most effective drugs at converting the gene expression profile from an EWS/FLI “active” profile to an “inactive” profile. As such, cytarabine appeared capable of mimicking blockade of EWS/FLI fusion oncoprotein production.

The gene-expression screening approach described in the manuscript will only identify compounds that produce the “EWS/FLI-off” profile as a mechanism of action against Ewing sarcoma. Other known effective compounds for this disease almost certainly utilize other mechanisms to elicit anti-tumor response. That having been said, doxorubicin, cyclophosphamide, and etoposide were also tested in this model. Doxorubicin produced the “EWS/FLI-off” profile, while cyclophosphamide and etoposide did not.

The Stegmaier group then further explored the effects of cytarabine on Ewing's sarcoma cells. EWS/FLI positive cell lines were treated with cytarabine for six days. Cytarabine at very low doses was again shown to decrease Ewing's sarcoma cell growth. Cytarabine appears to stimulate death in these cells. Cytarabine also reduces the amount EWS/FLI fusion oncoprotein produced by these cells.

Additional work has focused on the effect of cytarabine in mouse models of Ewing's sarcoma. One group evaluated a single dose of cytarabine in a mouse model of Ewing's sarcoma.¹¹ Compared to mice treated with placebo, this single dose of cytarabine did not reduce tumor volume. Due to the properties of this drug, though, a single dose of cytarabine may not be effective.

Another group has treated mice with Ewing's sarcoma tumors with cytarabine for 4 days. Once adequate tumors developed, mice received either placebo or cytarabine by injection into the abdomen once daily for four days. By seven days after the start of treatment, tumor activity increased 3-fold in placebo treated mice, while tumor activity decreased 6-fold in cytarabine treated mice. These results indicate that cytarabine demonstrates efficacy against both Ewing's sarcoma cells in culture and Ewing's sarcoma mouse models.

Clinical Experience with Cytarabine in Ewing's Sarcoma

No group has formally evaluated cytarabine for the treatment of Ewing's sarcoma or soft tissue PNET. Instead, two patients with this disease have been included in general studies of cytarabine in patients with a variety of solid tumors. For example, an adult with refractory Ewing's sarcoma received cytarabine in combination with mitoxantrone as part of a larger study.¹² This patient did not respond to therapy. A Pediatric Oncology Group study investigated high dose cytarabine (3 grams/m²/dose) in children with solid tumors.¹³ This trial included one patient with Ewing's sarcoma for whom response data is not available. We have found no other published reports of patients with Ewing's sarcoma receiving cytarabine. Interestingly, both of these patients received only two days of cytarabine, which may have provided inadequate exposure to this drug.

At least two groups have included patients with Ewing's sarcoma in studies of gemcitabine, a drug that is related to cytarabine.^14,15 A total of five patients with Ewing's sarcoma were treated on these two trials. One patient’s tumor remained stable while receiving gemcitabine. The other four patients’ tumors grew despite treatment with gemcitabine. The Sarcoma Alliance for Research through Collaboration (SARC) group is studying gemcitabine combined with another drug for patients with bone sarcoma. The results of this study are not yet available.

Overview of a Phase II Trial of Cytarabine for Ewing's Sarcoma

The Children’s Oncology Group (COG) has initiated a phase II trial of cytarabine for the treatment of Ewing's sarcoma. The goal of this study is to determine the response rate of cytarabine in patients with Ewing's sarcoma whose tumor has not responded to therapy or has recurred after completing therapy. This trial, known as AEWS0621, will be open to patients with relapsed or refractory Ewing's sarcoma or soft tissue PNET. Eligible patients must have measurable disease in addition to standard baseline bone marrow, liver, and kidney function. This trial will include patients less than or equal to 30 years of age at the time of study entry.

Patients on study will receive cytarabine 500 mg/m²/dose as an intravenous infusion every 12 hours for 5 days. This treatment will be repeated every 21 days for a total of two cycles before repeating scans to determine response to treatment. In the absence of tumor growth, patients will receive up to a total of 11 cycles on study. Due to the frequency of cytarabine administration, most patients will likely require hospitalization for five days at the start of each cycle.

This trial utilizes intermediate dose cytarabine. This dose was chosen for several reasons. A dose of 500 mg/m² cytarabine would be expected to achieve optimal blood levels of the drug.^16-18 This blood level far exceeds the levels needed to slow Ewing's sarcoma cell growth in laboratory studies. Others have demonstrated that this blood level results in optimal production of the active form of cytarabine.¹⁶ Although cytarabine is rapidly removed from the blood, for much of the interval following cytarabine administration on AEWS0621, blood levels are expected to remain above those required in laboratory studies.⁴ In addition, the half-life of the active form of cytarabine is several hours, suggesting that the active form of cytarabine will be present at adequate levels using this intermediate dose.¹⁶ Experience suggests that the side effects with this regimen will be reasonable. The selected dose will also likely avoid many of the complications associated with high dose cytarabine, including eye irritation and cerebellar toxicity.

High-dose cytarabine (typically considered doses > 1 gram/m²/dose) carries additional toxicities beyond those experienced with lower doses of cytarabine. Patients may develop a chemical conjunctivitis, thought to be due to high concentrations of cytarabine secreted in the tears following high-dose therapy. This chemical conjunctivitis can be effectively prevented using corticosteroid or normal saline eye drops during treatment with high-dose cytarabine. Cerebellar toxicity is a more serious consequence of high-dose cytarabine. Cerebellar toxicity is often heralded by nystagmus and ataxia after several days of high-dose therapy. If unrecognized and treatment continues, symptoms may progress and be irreversible. In the Ewing's sarcoma trial, an intermediate dose of cytarabine is being used that will likely avoid these additional toxicities seen with high-dose cytarabine.

In addition to assessing response to cytarabine based on radiology studies, AEWS0621 will also measure peripheral blood EWS fusion genes in patients being treated with cytarabine. Previous groups have measured Ewing's sarcoma tumor cells in the peripheral blood by measuring EWS fusion genes.^19-22 Detection of EWS fusion genes in the peripheral blood or bone marrow may predict tumor recurrence.^23-25 Whether decreases in peripheral blood EWS fusion genes correlate with decreases in tumor size remains less clear.^23,26 In AEWS0621, patients will have peripheral blood drawn to measure EWS fusion genes prior to starting cytarabine, at the start of every other cycle of therapy, and at the time of disease progression. The goal of this study is to determine whether decreases in peripheral blood EWS fusion genes correlate with response to cytarabine.

A second laboratory study included in AEWS0621 involves gene expression profiling of tissues obtained at initial diagnosis of Ewing's sarcoma. Previous work has demonstrated that gene expression patterns may predict response to specific therapies in other types of cancer.^27-29 In AEWS0621, the gene expression profile will be determined from available diagnostic samples and correlated with conventional disease response.

Summary

Patients with relapsed or refractory Ewing's sarcoma often have a poor outcome with conventional therapy. Laboratory studies suggest that cytarabine may be effective for these patients by affecting the EWS/FLI fusion oncoprotein that is characteristic of this disease. Clinical evidence regarding the utility of cytarabine in patients with Ewing's sarcoma is lacking. A current COG study, AEWS0621, is formally evaluating the efficacy of intermediate dose cytarabine for this disease. If successful, cytarabine will be the first agent to successfully target the EWS/FLI oncoprotein in the treatment of patients with Ewing's sarcoma.

More Detailed Treatment

A more detailed treatment of the issues discussed in this article is to appear in: Stegmaier K, Wong JS, Ross KN, Chow KT, Peck D, et al. (2007) Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma. Public Library of Science MEDICINE (PLoS Med). In Press.

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