—  SYMPOSIUM #16  —

History of Bone Tumor Pathology
Moderators: K. Krishnan Unni and Franco Bertoni

Section 4 - Diagnosis and Treatment of Ewing Sarcoma

Andrew G. Huvos


Introduction
Ewing sarcoma is a malignant tumor of small round cells arising in bone. The exact nature and origin of the cells has been a matter of much debate. [1, 2] Ewing, who is credited with bringing attention to the disorder, preferred to call it "diffuse endothelioma of bone." [3] Others believed it was derived from skeletal mesenchyme. [4] Still others proposed that it was merely a form of metastatic neuroblastoma. As a result of recent cytogenetic and molecular studies, Ewing sarcoma is now thought to be a member of a family of tumors that includes primitive neuroectodermal tumors (PNET), peripheral neuroepithelioma, Askin's tumor of the chest wall, and extraosseous Ewing sarcoma. [2] These tumors probably arise from a neuroectodermal stem cell rather than mesenchymal tissue, but definitive proof is still lacking.

Ewing sarcoma is one half to one third as common as osteogenic sarcoma in the U.S., but among patients less than 15 years of age, Ewing sarcoma is nearly as common as osteogenic sarcoma. [5] This reflects the fact that Ewing sarcoma has a sharper peak incidence in younger patients than osteogenic sarcoma and is rare beyond the third decade. However, the development of new cytogenetic and molecular diagnostic tools may allow the detection of more cases in the elderly that previously were ascribed to poorly differentiated round cell tumors.

The disease usually affects Caucasians and is distinctly uncommon in blacks and Asians. The male to female ratio is approximately 3:2. [6] The pelvis and femur are favored locations, but many other bones may be involved, including the humerus, tibia, and fibula. Primary soft tissue involvement has a predilection for the paravertebral muscles.

The etiology is related to a chromosomal translocation. In over 90% of cases, there is a reciprocal t(11;22)(q24;q12) translocation that results in a fusion of the EWS gene to the Fli1 gene. In approximately 5% of cases there is a 21;22 translocation that fuses the EWS gene to the ERG gene, and in rare cases the EWS gene may be fused to other genes such as the E1A gene and the ETV1 gene. [7, 8] The chimaeric proteins function as aberrant transcription factors. It is believed that the fusion proteins activate and/or repress a set of genes which result in neoplastic transformation of the cell, but the critical target genes have yet to be identified.

Clinical Manifestations
Most patients complain of pain and swelling at the affected site. Growth of the tumor is rapid, and symptoms are typically present for only weeks to months. In most cases, a substantial firm mass is present, and its sudden appearance and enlargement may cause alarm in the patient. The presentation can simulate acute osteomyelitis, and some patients have constitutional symptoms of fevers, malaise, and lethargy. Pathologic fractures occasionally occur.

Radiographic Differential Diagnosis
Ewing sarcoma has protean radiographic manifestations and is notorious for its ability to masquerade as other disorders. The most well-known finding — onionskin formation — is not consistently present. Moreover, it is not a unique attribute and can be produced by numerous other diseases, including osteomyelitis, Langerhans cell granuloma, and osteogenic sarcoma. Onionskin formation is one form of reactive periosteal bone formation. Other forms include the sunburst pattern ("hair-on-end" bone) and Codman's triangle. In all of these variations, the new bone is not made by the tumor but by the periosteum, which is elevated off the cortical bone by a rapidly expanding mass. In Codman's triangle, the mass erodes through the central portion of the onionskin laminations, leaving only the triangular ends on the bone. In a sunburst pattern, the central portion is filled in by spicules of new bone radiating outward perpendicular to the shaft.

Ewing sarcoma usually produces an ill-defined, lytic defect that permeates up and down the medullary canal, giving the bone a moth-eaten appearance. However, in approximately 10% of cases, the tumor may have a predominantly blastic appearance as a result of exuberant reactive bone formation. This can cause it to be confused with osteogenic sarcoma, particularly the small cell variant. [9]

An important clue that suggests the possibility of Ewing sarcoma is the presence of a large soft tissue mass adjacent to the bone. This may be subtle and difficult to appreciate on plain radiographs but becomes apparent with CT or MRI scans. In certain bones such as the pelvis, periosteal reaction is often absent radiographically, and the soft tissue mass becomes more important to making the diagnosis.

Laboratory tests may show leukocytosis with a left shift, and the erythrocyte sedimentation rate may be elevated. These findings, along with the history, examination, and radiographs, can easily deceive the clinician into thinking that the diagnosis is osteomyelitis. The serum lactate dehydrogenase (LDH) is important to note since it is correlated to the disease burden, and it has prognostic importance.

Pathologic Differential Diagnosis
The tumor is composed of sheets of small round blue cells with hyperchromatic nuclei. There is scant cytoplasm and little extracellular matrix. The cells are usually glycogen positive. Neural markers are occasionally present, and these can include neuron-specific enolase, CD57, neurofilaments, S100 protein, and Homer-Wright rosettes. [10]

Ewing sarcoma may be morphologically indistinguishable from other small round cell tumors, such as lymphoma of bone and metastatic neuroblastoma. Differentiation from these other entities has been facilitated in recent years by the development of the monoclonal antibodies HBA71 and O13 against Ewing sarcoma. [11] The antibodies recognizes the p30/32 MIC2 protein, which was originally described as a cell surface marker of T lymphocytes. [12] The function of the MIC2 protein is not completely understood, but in T cells it is thought to be involved in cell adhesion. Immunohistochemistry with the O13 antibody is positive in most cases of Ewing sarcoma. [13, 14, 15, 16]. In a large study of 244 cases, the antibody was found to be 91% sensitive. [17] However, the antibody is not 100% specific, and it occasionally cross-reacts with lymphomas and other tumors. [18] This is not surprising since the MIC2 protein has been detected by another antibody 12E7 on most lymphoblastic lymphomas and T cell acute lymphocytic leukemias. [18] Other tumors that occasionally bind O13 include astrocytomas, neuroectodermal tumors, mesenchymal chondrosarcoma, embryonal rhabdomyosarcomas, and carcinomas. [13, 15] It is notable that neuroblastomas have not been found to react with O13.

The development of reverse transcriptase-polymerase chain reaction (RT-PCR) has also aided the diagnosis of Ewing sarcoma by facilitating the detection of specific chromosomal translocations. [19] RT-PCR is a powerful test, and one study reported 100% sensitivity and specificity in detecting the 11;22 translocation. [20] However, other tumors besides Ewing sarcoma occasionally possess the same translocation, and one study found it in two polyphenotypic tumors and two mixed rhabdomyosarcomas. [21] Thus, like the O13 antibody, RT-PCR cannot be relied upon exclusively to make the diagnosis, and it is still important to consider all of the histologic and clinical data.

Clinical Staging
The staging system for Ewing sarcoma differs from the system used for most sarcomas of bone. This seems appropriate since the histogenesis of Ewing sarcoma may not be mesenchymally derived and the clinical behavior seems to differ from most sarcomas. Enneking suggested four stages: stage I, solitary intraosseous tumor; stage II, solitary tumor with extraosseous extension; stage III, multicentric skeletal involvement; and stage IV, distant metastases. [22]

The staging system reflects the propensity of Ewing sarcoma to disseminate widely and early in the course of the disease. Lungs and other bones are the usual sites of metastases. A distinctive feature of Ewing sarcoma is its predilection for bone marrow involvement, which is uncommon in other sarcomas. [23] The finding carries a poor prognosis, and Meyers et al found no survivors when it was present. [24] Bone marrow biopsy should be part of the standard staging studies for Ewing sarcoma.

Treatment
Treatment of the primary tumor consists of radiation therapy, surgery, or a combination of both modalities. Historically, radiation therapy has been used most often. It was recognized by Ewing and others that the tumors are sensitive to radiation. However, survival with radiation alone was less than 10%, reflecting the presence of microscopic disseminated disease at the time of diagnosis. Surgery, which usually involved amputation, produced equally poor results, and consequently, many authors condemned the use of surgery for Ewing sarcoma. [25, 26]

Radiation has produced rates of local control ranging from 60-90%. There are a number of explanations for the wide variation in results. Techniques of radiation therapy have improved over time. Radiation in older trials may have been hampered by inadequate equipment as well as poor imaging techniques. The advent of CT and MRI has led to more accurate depiction of the tumor and better selection of radiation fields. Yet despite the advances in radiation therapy, a number of recent reports have continued to show disappointing results. A POG study found only 76% local control at three years [27] while a CESS study reported only 77% local control at five years. [28] Recurrences have been noted to occur within radiation fields, and autopsies have demonstrated viable malignant cells in irradiated tumors. Tepper et al found live neoplastic cells in 11 of 28 primary tumors, [29] and Telles et al found recurrent disease in 13 of 26 tumors. [30] A certain amount of selection bias was inherent in these autopsy studies since only patients who failed treatment were analyzed. Nevertheless, the studies clearly demonstrate that many tumors are not completely eradicated by radiation.

Local recurrence after radiation is associated with a number of factors. The size of the tumor is important. Large tumors (greater than 100 ml in volume) are much more likely to harbor a focus of radioresistant cells than small tumors. [28] The location of the tumor is also important. Central and pelvic tumors have much higher rates of local recurrence than distal tumors. [31] This may reflect the difficulty in achieving adequate radiation doses around vital organs.

The response to chemotherapy also affects the likelihood of local recurrence. It was first noted that patients who received chemotherapy had lower overall rates of local recurrence than patients who did not receive chemotherapy. [32, 33] Arai et al subsequently found that patients who responded well to chemotherapy had significantly better local control than patients who responded poorly. [34] In this study, patients who had an objective response to chemotherapy and a tumor less than 8 cm long achieved 90% local control, despite being given a low radiation dose (35 Gy). Patients who had an objective response to chemotherapy and a tumor more than 8 cm long were given a high radiation dose (50-60 Gy), but these patients obtained only 52% local control. Finally, patients who failed to show a response to chemotherapy were also given a high dose (50-60 Gy), but had only 17% local control.

In addition to improving local control with radiation, effective adjuvant chemotherapy has made limb-sparing surgical excision possible. In fact, surgery may be a superior alternative to radiation treatment. The most cogent argument in favor of surgery is that it can remove a focus of radioresistant and chemoresistant cells that may reside within a large tumor. Another compelling argument is that patients who undergo radiation are at a substantial risk for developing a second primary tumor, particularly osteogenic sarcoma. [35, 36, 37, 38, 39, 40, 41] The cumulative risk increases with time and has been estimated to be 8.6% at 20 years, with a mean latency of 7.6 years. [35] Patients who receive a radiation dose of 60 Gy or greater seem to be at greatest risk. [35, 42, 43]

Many clinicians have objected to surgery on the basis of its being invasive, destructive, and disfiguring. They have preferred radiation since it seems to be non-invasive and limb-preserving. This viewpoint may have had some merit in the past, but it is no longer completely valid. Surgical reconstructive techniques have improved considerably, and function after limb-sparing surgery is significantly better now than in the past. Surgery should no longer be reserved for "expendable" bones, such as the ribs, clavicle, and fibula. [44] With modern surgical techniques, there are few, if any, truly non-expendable bones, and reconstructive options exist for essentially all anatomic sites. Furthermore, it is worth emphasizing that radiation produces a significant amount of damage to normal tissue and can potentially cause serious functional impairment. Complications of radiation include skin atrophy and breakdown, fibrosis of muscles, contracture of joints, vasculitis, neuropathy, growth plate arrest, limb length inequality, osteonecrosis, and pathologic fractures. [45, 46]

Although it is important to compare radiation and surgery with respect to functional outcome, the focus of the current debate should center on oncologic outcome. At present, the published data seems to favor surgery but is not entirely conclusive. Several studies have shown that surgery produces a higher rate of local control. Bacci et al found 36% local recurrence with radiation alone compared to 8% local recurrence with either surgery alone or surgery with radiation. [36] Ozaki reported 15% local recurrence with radiation therapy alone compared to 4% local recurrence with surgery alone and 4% with surgery and radiation therapy. [47] The main criticism of these and other studies is that they were not randomized trials, and there may have been a selection bias towards surgical excision of relatively smaller and more distal tumors.

There is only limited data pertaining to large, centrally located tumors, which carry the worst prognosis. [36, 48, 49] Some have recommended radiation therapy for these sites because it is difficult to obtain negative surgical margins, and the rate of complications is high. However, radiation treatment is also difficult in central locations because of their proximity to vital organs, and these tumors are precisely the ones most likely to recur after radiation.

There is conflicting data on whether surgery may have a positive impact on pelvic tumors. Scully et al found that there was no difference in survival between radiation therapy and surgery for pelvic tumors. [50] The experience of the Rizzoli Institute was similar, and survival was not improved by the addition of surgery to radiation. [51]In contrast to these findings, several groups have reported more favorable results after surgical treatment of pelvic tumors. [52, 53, 54, 55] In the study from Memorial Sloan-Kettering, nine of 12 cases of pelvic tumors were treated surgically, and there were no local recurrences in these nine tumors. [53] At UCLA, Yang et al obtained 51% cumulative 5-year survival in patients who had surgical resection compared to 18% survival in patients who did not have surgery. [54]

Some have proposed that a combination of surgery and radiation be used to improve the rate of local control. Data on this subject is limited and confounded by a significant selection bias for patients that receive both radiation and surgery. Often these are the patients with the greatest perceived risk of recurrence — those with positive margins, large tumors, or contamination by previous surgery. This may be the reason that some investigators have not found an advantage to combining surgery with radiation. [47, 51] More encouraging results were found by Wunder et al at Memorial Sloan-Kettering. Among patients who had a complete en bloc resection, the relative risk of local recurrence was 3.9 for patients who did not receive radiation compared to those that did receive radiation. [56] All six patients with local recurrence in this series subsequently died of disease. The results at UCLA also seem to indicate a potential benefit of combined radiation and surgery in selected patients. [54] If indeed there is an increase in both local control and survival, the benefits of combining the two modalities may outweigh the increased risks of complications that would be expected from this aggressive approach. Proposals to conduct randomized trials of radiation versus surgery fail to consider that many patients may benefit from both modalities.

The issue of surgical treatment is further complicated by the choice of margins. Should the margin be outside of the original tumor, outside of the tumor after induction chemotherapy, or outside of the tumor after induction chemotherapy and radiation? Theoretically, it seems most prudent if the margin around the original tumor is chosen, but this requires sacrificing the most tissue. A margin outside of a tumor that has shrunk after induction chemotherapy and/or radiation is less optimal but may be chosen to spare important structures such as major nerves

Pathologic fractures, especially those occurring after radiation, poses another surgical dilemma. In the study by Terek et al, all femoral lesions treated initially with radiation eventually required surgical treatment, either as a result of recurrence or pathologic fracture which failed to unite. [57] Healing of these post-radiation fractures can occur but is often delayed. [58]

It is likely that no single therapeutic strategy will be ideal for all patients. Surgery may theoretically improve the chances for local control, but patients should be carefully selected, and the lesion must be resectable with adequate margins. In patients at high risk of local recurrence, surgery may be combined with radiation, but there may be an increase in wound complications. In the rare, unresectable lesion, radiation alone may be the only choice, but it is possible that after radiation and induction chemotherapy, the lesion may regress and become resectable.

Adjuvant therapy
The development of chemotherapy for Ewing sarcoma began in the 1960s, when phase II trials in patients with metastatic disease showed promise for cyclophosphamide, actinomycin D, vincristine, and other agents. Successful adjuvant chemotherapy for patients with non-metastatic disease was reported by Hustu in 1968, who treated five patients with vincristine and cyclophosphamide. [59] It may be more than mere coincidence that the chemotherapy was adapted from protocols for neuroblastoma, and the neurotoxic agent vincristine was used.

The first Intergroup Ewing Sarcoma Study (IESS-1) began accrual of patients in 1973. [60] This involved 3 major study groups (CCSG, SWOG, and CALBG) and 84 participating institutions. The chemotherapy was based on vincristine, actinomycin D, and cyclophosphamide (VAC), and patients were randomized to three groups: VAC alone, VAC plus doxorubicin (VACA), and VAC plus whole lung irradiation. Radiation was used to treat the primary tumor. The IESS-1 study firmly established the effectiveness of adjuvant chemotherapy. Patients that received VAC plus doxorubicin had a 5 year relapse-free survival of 60%, which was far superior to any historical control. Patients that received only VAC had a survival of only 28%, which demonstrated the effectiveness of doxorubicin. Survival with VAC + whole lung irradiation gave intermediate results with survival of 53%, which was better than VAC alone, but was not as effective as the VACA combination.

In the follow-up IESS-2 trial, intermittent high dose chemotherapy was shown to be more effective than continuous moderate dose chemotherapy. [61]Dose intensity, especially that of doxorubicin, was found to be an important factor. [62] A 5 year disease-free survival of 68% was achieved in the intermittent high dose chemotherapy group, but there was greater cardiotoxicity and one cardiac-related death. [61]

Subsequent trials have corroborated the findings of IESS-1 and confirmed the effectiveness of VACA. The results of selected major trials are shown in Table 4. The CESS 81 study obtained 55% disease-free survival at 69 months [28] while the Rizzoli group found 54% disease-free survival at 5 years. [36] Hayes et al used these four agents in a somewhat different protocol and reported 3 year disease-free survival of 82% for tumors less than 8 cm and 64% for tumors greater than 8 cm. [63]

Rosen at Memorial Sloan-Kettering reported a 10 year experience with a series of 67 consecutive patients from 1970-80. [53] The protocols evolved during this period from T2 to T6 to T9. The T2 protocol consisted of VACA, while the T6 and T9 protocols employed doxorubicin, cyclophosphamide, high dose methotrexate, BCD (see osteogenic sarcoma above), and BCNU (T6). Although excellent results were reported with 79% disease-free survival at 2 years, which was stable to 5 years, the interpretation of the study is complicated by the variety of agents and protocols. Many of the early patients received extremely high doses of doxorubicin in the range of 700-900 mg/m2, which is beyond the doses allowed under current protocols. In a follow-up study of patients that received T9 and T11 chemotherapy at the same institution, Meyers found 53% event-free survival at 5 years. [24] This study, however, included patients with metastatic and relapsed disease.

There is currently much interest in the use of ifosfamide and etoposide (VP16), which have been shown to be effective in phase II trials. [64, 65, 66, 67, 68] However, addition of these agents to the traditional four agent regimen VACA may produce only a small increase in survival. A large study from the Rizzoli Institute found only a 4% increase in disease-free survival, which was not statistically significant. [67, 70] Preliminary data from a recent large, randomized CCG/POG study of 398 patients showed more encouraging results, and the five year event-free survival was increased significantly from 52 to 69%. [68]

In recent years there has been an effort to stratify patients into high risk vs. standard risk categories. The definition of high risk varies, but usually includes metastatic cases (lung, bone, and/or bone marrow), relapsed cases, and primary tumors in unfavorable locations. The latter category is comprised of axial and pelvic tumors, but many investigators have also included "proximal" tumors in the humerus and femur. Large tumors have also been included in high risk categories, with criteria being either >8 cm greatest dimension or >100 ml volume. [63, 71]

It seems that if all of the above tumors are considered "high risk" then only a small percentage of cases would qualify as "standard risk." Not all of the high risk cases are at similarly high risk. The prognosis in axial or pelvic locations is worse than the humerus or femur. Likewise, the prognosis for patients that relapse while on chemotherapy is bleak, whereas the prognosis for patients that present with metastatic disease may be somewhat better. [24, 72, 73, 74] Cangir et al, reporting the IESS experience, found that the 5 year survival was 30% for patients that presented with metastases (ie, patients who did not relapse during treatment). [72] Sandoval similarly found an overall survival of 35% for cases that presented de novo with metastases. [73] The type (location) and timing of metastases appear to be important. Meyers found no survivors in patients with bone marrow involvement or patients that developed metastases while on chemotherapy. [24] These considerations should be kept in mind when reviewing the literature on high risk cases. The results that pertain to one set of "high risk" patients may very well not apply to another group.

In contrast to osteogenic sarcoma and other sarcomas, there has not been much success with surgical resection of metastases. Heij reported that all 12 patients who underwent thoracotomy for metastases eventually died of the disease. [75] It is probable that the behavior of Ewing sarcoma differs from osteogenic sarcoma, and Ewing sarcoma has a much greater propensity for widespread microscopic dissemination.

Bone marrow transplant and stem cell rescue have been used for relapsed and high risk cases. [52, 76, 77, 78]. This approach seems especially applicable to cases of bone marrow metastases, which might respond to bone marrow ablation by total body irradiation and/or high-dose chemotherapy. Total body irradiation has a additional appeal since Ewing sarcoma is radiosensitive. However, it should be recalled that the responses below 30 Gy were unpredictable, and a significant percentage of cases do not respond well to radiation therapy even at high doses. [34]

Cornbleet et al reported in 1981 on 3 patients with refractory Ewing sarcoma treated with high dose melphalan and autologous bone marrow rescue. [79] Two patients had a complete response and survived free of disease for 12 and 13 months. One patient had a partial response. A more extensive study was performed at the NCI, where patients were treated with VACA, radiation therapy for local control, 8 Gy total body irradiation, and autologous bone marrow rescue. [76] Early results were impressive with 30/31 complete responses, but long-term results were less encouraging. Of the 13 metastatic cases, 9 relapsed and the projected 6 year survival rate was only 10%. The non-metastatic, high-risk cases fared better with only 5 of 18 cases relapsing. Burdach et al reported more optimistic results in a group of 17 patients with metastatic disease (7 new cases, 10 relapsed cases). [78] Treatment consisted of induction chemotherapy, local treatment, myeloablation with melphalan/etoposide +/- carboplatin, 12 Gy total body irradiation, and finally bone marrow or stem cell rescue. The 6 year relapse-free survival was 45%.

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