lmmunohistochemical Expression of Neuron-Specific Enolase and Leu 7 in Ewing’s Sarcoma of Bone

ALFRED0 PINTO, MD, LESTER H. GRANT, MD, DPHIL, F. ANN HAYES, MD, MICHAEL J. SCHELL, PHD, AND DAVID M. PARHAM, MD

Neural features have been documented in a series of small round cell tumors of bone, previously classified as Ewing’s sarcoma of bone (ESB), using light microscopic, ultrastructural, immunohistochemical, and in vitro techniques. To date, correlation of the presence of these features in ESB with clinical outcome has not been performed. The authors investigated the clinical relevance of positivity to antibodies against neuron-specific enolase (NSE) and Leu 7 (HNK 1) using the avidin-biotin complex technique in 40 cases of ESB seen at St. Jude (Memphis, TN) during the period 1968 to 1986. Twenty-three cases (58%) were positive for NSE and/or Leu 7. The median disease-free survival of patients with localized tumors and NSE and/or Leu 7 positivity was 3.8 years compared to a median disease-free survival of 1.6 years for those without these markers and localized disease. Using the log-rank test, a statistically significant difference was shown in these two groups of patients (P = 0.049). The authors conclude that in this relatively small series ESB commonly expresses NSE and/or Leu 7 and that the presence of these markers is of no prognostic significance overall, but that in patients with localized ESB the presence of NSE and/ or Leu 7 by immunostaining may be a favorable prognostic indicator.

Cancer 64:1266-1273, 1989.

INCE 1921, when James Ewing first described a diffuse S endothelioma of bone,’ several studies have at- tempted without success to clarify the histogenesis of Ew- ing’s sarcoma (ES). In recent years, with the use of new methodologies, such as histochemistry, monoclonal an- tibodies, and molecular genetics, it has become clear that ES represents a primitive mesenchymal neoplasm which has the potential of multidirectional differentiation.2 Neural differentiation has been induced in Ewing’s tumor cell lines3 and neural and neuroectodermal antigens have also been detected in ES cell lines4,’ and in tissue sec- t i o n ~ . ~ , ~ A specific translocation, the t( 1 1 ;22), has been found in both ES and in peripheral primitive neuroec-

Presented in part at the United States and Canadian Academy of Pathology (United States-Canadian Division of the International Acad- emy of Pathology), Washington, DC, February 28-March 4, 1988.

From the Departments of Pathology and Laboratory Medicine, He- matology-Oncology, and Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee; and the Departments of Pathology and Pediatrics, University of Tennessee, Memphis, Tennessee.

Supported by the American Lebanese-Syrian Associated Charities (ALSAC) and by the Alberta Children’s Hospital Foundation.

The authors thank Ms. Hallie Holt and Ms. Vicki Denison for their technical skills; Ms. Alice Slusher for preparation of immunohistochem- i d and electron microscopic materials; and Ms. Vivian Turner for typing the manuscript.

Address for reprints: David M. Parham, MD, Department of Pathology and Laboratory Medicine, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38 101.

Accepted for publication March 18, 1989.

todermal tumors (PNET).’-’* At the molecular level a profile of protooncogene expression supports the evidence of a genetic and biological relationship between ES and PNET.’ ’ Ewing’s sarcoma of bone (ESB), ES of soft tissue, PNET, Askin’s tumor, and ES with neural differentiation all appear to represent a group of tumors which share common antigenic profiles as well as cytogenetic and pro- tooncogene expression. This group of tumors may rep- resent a common entity with different degrees of differ- entiation. Whether the presence of some of these markers influences biological behavior of the tumor is currently unknown. In an effort to understand better the significance of the presence of neural markers in ES of bone, we per- formed a retrospective analysis of 40 cases diagnosed as ESB and treated as such.

Materials and Methods

Tissue Specimens

One hundred thirty-nine cases of ESB were retrieved from the files of Surgical Pathology at St. Jude Children’s Research Hospital (Memphis, TN). The cases were di- agnosed during the period from 1968 to 1986. Forty cases were selected on the basis of adequate availability of par- affin-embedded nondecalcified tissue. In 16 cases ade- quate, well-fixed electron microscopic material was avail- able for examination.

1266

No. 6 NEURAL MARKERS IN EWING’S SARCOMA * Pinto et al. 1267

Light Microscopic Study

Hematoxylin-eosin-stained sections of all 40 cases were examined by two observers (D.P. and A.P.). Periodic acid- Schiff-stained sections were available in most cases. The diagnosis was confirmed after excluding other small round cell sarcomas. Several sections were examined in each case. In each case the slides selected for immunostaining were fixed in formalin and were not decalcified.

Immunologic Staining A panel of antibodies was used in all cases and included

polyclonal anti-neuron-specific enolase (NSE) (Dako Corp., Santa Barbara, CA, dilution 1:200); Leu 7 (HNK 1) (courtesy of Dr. T. Triche, dilution 1:40); polyclonal anti-desmin (Dako, dilution 1 : 100); anti-leukocyte com- mon antigen (LCA) (Dako, dilution 1 : 100); polyclonal anti-creatine kinase MM (CKMM) (Cambridge Medical Diagnostics, Inc., Cambridge, MA, dilution 1: 100); nor- mal rabbit serum (Dako); and purified mouse ascites pro- tein (MOPC 21) (Sigma, St. Louis, MO). On a selected number of NSE-positive cases, immunostaining was per- formed using monoclonal anti-synaptophysin (Boeringer- Mannheim, Indianapolis, IN, undiluted). For all anti- bodies except Leu 7, immunoperoxidase staining was performed on paraffin sections using the avidin-biotin- peroxidase complex (ABC) method.” The indirect im- munoperoxidase technique13 was used to demonstrate Leu 7. Positive controls for each antibody stain were repre- sented by sections of normal peripheral nerve, striated muscle, pancreas, lymph node, and ganglion cells in large bowel. Negative controls consisted of substituting MOPC 21 for monoclonal antibodies and normal rabbit serum for polyclonal antisera. Interpretation of the staining was made by two observers (D.P. and A.P.) independently. When disagreement occurred, the slides were reviewed in a dual-headed microscope and a consensus opinion was reached. We scored the results as follows: -, no staining; +, staining in less than 10% of the tumor cells; ++, stain- ing in 10% to 50% of the tumor cells; +++, staining in greater than 50% of the tumor cells.

Electron Microscopic Exumination Electron photomicrographs and embedded blocks were

available for ultrastructural examination in 28 cases. In 16 cases the material was considered optimal for exami- nation because of good fixation and availability of Epon- embedded blocks. Before embedding in Epon, the tissue was previously fixed in phosphate-buffered glutaraldehyde 2.5% (pH:7.0), washed in 0.1 mol/l phosphate buffer, postfixed in 1 % osmium tetroxide, dehydrated in graded solutions of alcohol acetone, immersed in proxylene oxide, and embedded in Epon. Sections were cut in an MT-5000 Sorval Ultra-microtome (Dupont-Sorval, Wilmington,

DE) and double stained with uranyl-acetate and lead ci- trate. Sections were examined in a Phillips 301 electron microscope (Phillips Electronic Instruments, Inc., Mah- wah, NJ).

Clinical Aspects

The clinical information regarding diagnosis, therapy, and follow-up was obtained from the medical records at St. Jude Hospital. Before 1978, patients were treated with vincristine, cyclophosphamide, dactinomycin -t j - Adri- amycin (doxorubicin), weekly or every 2 weeks for 18 to 24 months. After 1978, these four drugs were delivered in a sequential fa~hion.’~ Patients with rib primaries and pleural effusions but with no objective evidence of separate pleural masses were not considered as having metastatic disease. Each patient was assigned a clinical response cat- egory using the following criteria. Complete response (CR): complete resolution of all soft tissue tumor (primary and metastatic sites) by computed tomography (CT) scan with improvement in bone lesions by radiographs, bone, and gallium scans, and absence of tumor cells on bone marrow examination; partial response (PR): >50% regression of all soft tissue tumor by CT scan; and no response (NR): 4 0 % regression of soft tissue tumor by CT scan or progressive disease occurring during the in- duction period regardless of any interval response.

Survival intervals were calculated from the date of di- agnostic biopsy to the most recent follow-up date. Disease control interval was calculated from date of diagnostic biopsy to the date when progressive or recurrent disease was documented. Survival distributions were estimated by the product-limit method of Kaplan-Meier. Differences between survival distributions were analyzed by the log- rank test.15

Results

Light Microscopic Findings The histologic patterns were classified as previously de-

scribed by the Intergroup Ewing’s Sarcoma Study.16 Twenty-eight of the cases revealed a predominantly diffuse pattern, eight showed a lobular pattern, and in four cases the pattern was predominantly filigree. Cytologically the individual cells displayed a round or ovoid nucleus with thin nuclear membrane and finely distributed chromatin pattern as well as scanty and ill-defined cytoplasm. Sig- nificant variation in the size of the nucleus with the pres- ence of distinctive nucleoli was seen in five cases which were regarded as atypical variants. No angiomatoid vari- ants were found among our cases. Apoptotic rosettes, pseudorosettes, and abortive rosettes were frequently seen. Well-formed rosettes with a central fibrillary core (Homer- Wright type rosettes [HWR]) were found focally in five cases (Fig. 1). In one case HWR and true rosettes with an

1268 CANCER September 15 1989 Vol. 64

FIG. 1. Ewing’s sarcoma of bone. Rosette with fibnllary core (H & E, X200).

empty central lumen (Flexner-type rosettes) were observed (Fig. 2).

Microscopic soft tissue invasion was seen in 23 cases and intramyofiber muscle invasion was found in five cases.

Immunoperoxidase Staining

The results of immunoperoxidase staining are shown in Table 1. Neuron-spec.ific enolase was positive in 19 cases, with the staining confined to the cytoplasm. The pattern of staining was focal and the intensity was weak to moderate (Fig. 3). According to the grading described above, 14 cases were scored as +; four cases as 2+; and one case as 3+. Leu 7 was positive in 12 cases. The staining was also localized in the cytoplasm and of moderate in- tensity (Fig. 4). Ten cases were scored as + and two cases as 2+. Eight cases were positive for both NSE and Leu 7, and 23 cases were positive for either NSE or Leu 7. The reactivity of staining was always abolished when the pri- mary antibody was replaced by normal rabbit serum or by MOPC 2 1. Three of the five cases with HWR showed

FIG. 2. Ewing’s sarcoma of bone. Rosette with empty central lumen (H & E, XZOO).

TABLE 1. ESB Immunostaining With NSE and Leu 7

Leu 7+ Leu 7- ’Total

NSE+ 8 11 19 NSE- 4 17 21

Total 12 28 40 ~

ESB: Ewing’s Sarcoma of bone; NSE: neuron-specific enolase.

reactivity with either NSE or Leu 7. Staining with LCA, desmin, and CKMM was negative in all cases. No cor- relation was found between the predominant pattern and NSE and/or Leu 7 staining (data not shown). All five cases regarded as atypical variants showed focal positivity with either NSE or Leu 7. Antisynaptophysin staining was per- formed on eight cases having positive NSE staining rang- ing from 1 + to 3+. Only one case, having a 3+ NSE stain, showed a definite focal 1 + synaptophysin positivity (Fig. 5). Two additional cases gave a weak diffuse positivity which was difficult to distinguish from background stain- ing due to the low dilution used in the procedure. The remaining five cases appeared negative.

Electron Microscopic Results

Special care and time were taken to determine the pres- ence or absence of microtubules, dendritic processes and dense core granules. We scored the case as positive for neural differentiation when two or more of these features were present. Cytoplasmic processes were found in nine cases with five showing well-developed processes. Dense core granules were found in the cytoplasmic processes in four cases and in the cytoplasm in one case. In only one case were typical dense core granules detected (Fig. 6) . The size of the granules ranged from 100 to 400 nm in diameter. The granules were scanty and no more than four granules per process were found. In two cases mi- crotubules were found in the perinuclear area and in the cytoplasmic processes. One case with HWR showed neural features with typical dense core granules. In the other cases with HWR, no electron microscopic (EM) material was available for examination. The majority of tumors showed a predominance of clear cells measuring from 10 to 18 pm with ultrastructural features typical of those previously described for ES (scanty cytoplasm, poorly developed cy- toplasmic organelles, and focal pools of glycogen) ( 1 41 16 cases). Scattered dark cells were observed in 13 cases.

Among the 16 cases with ultrastructural studies, ten cases were scored as negative and six cases were tentatively scored as positive. Among the EM-positive cases only one stained positively for NSE/Leu 7 immunoperoxidase stain. This case had typical dense core granules. In the other five cases with atypical dense core granules and ei- ther microtubules or dendritic processes, tentatively scored

No. 6 NEURAL MARKERS IN EWING’S SARCOMA - Pinto et a1 1269

FIG. 3. NSE staining in the cytoplasm of ES cells (ABC method, X200).

as positive for neural features, the staining with NSE and/ or Leu 7 was negative. The nature of the atypical dense core granules is uncertain in these cases; they probably represent primary lysosomes.

Clinicopathologic Correlations

The clinical and pathologic data are summarized in Table 2. Sixteen were female patients and 24 were male patients; 38 were white and two were black. The mean age of the patients at diagnosis was 14 years (range, 2-20 years).

Twenty-eight patients had a predominantly diffuse pat- tern: 1 1 are alive and 17 died of progressive disease. Eight patients had a lobular pattern: four are alive and four are dead. Four patients had a filigree pattern: two are alive and two are dead. No definite relationship between sur- vival and site of primary tumor was seen in our study (Table 2). However, it should be noted that all four pa- tients with a primary scapular tumor died of progressive disease, and that the one patient with a primary in a metacarpal bone is alive. All five patients with intramyo- fiber invasion died of progressive disease.

Response to Therapy

Seventeen of the 34 patients who achieved CR had NSE-positive and/or Leu 7-positive tumors. Seven pa- tients who later achieved CR presented with metastasis at the time of diagnosis; the remainder presented with localized disease. Additionally, one patient with a local- ized, NSE-positive and Leu 7-positive tumor in the scap- ula had a PR. Five patients, including two with metastatic disease at diagnosis, had NSE and/or Leu 7-positive tu- mors and were nonresponders.

Survival Analysis

Seventeen patients were alive at the time of this analysis. Three (one with positive NSE and/or Leu 7) had meta-

FIG. 4. Leu 7 staining in the cytoplasm of ES cells (indirect method, X200).

static disease at diagnosis and fourteen (ten with positive NSE and/or Leu 7) had localized disease. No statistically significant differences were observed in the overall survival when the group of patients with NSE and/or Leu 7-pos- itive tumors were compared to the group of patients with NSE/Leu 7-negative tumors (Fig. 7); however, this result is not unexpected, since the number of patients in this series is small. Disease control was analyzed in the 31 patients with localized disease. Five of the 18 patients with neural markers were alive and free of disease com- pared to none of the 13 patients with negative markers ( P = 0.049, Fig. 8). The median disease control interval for patients with localized tumors and positive markers was 3.8 years, whereas the median disease control interval for patients with localized tumors and negative markers was 1.6 years. Seven of nine patients with metastasis at diagnosis achieved CR but only three are alive currently, including one with an NSE-positive tumor. Tumor size was recorded before therapy in 19 patients. Among the 15 patients with tumors greater than 8 cm, nine are alive (five with NSE-positive and/or Leu 7-positive markers)

FIG. 5. Focal synaptophysin staining in the cytoplasm of a cluster of ES cells (ABC method, X200).

1270 CANCER September 15 1989 Vol. 64

FIG. 6. Electron micrograph of Ewing's sarcoma cells with cytoplasmic processes and rare dense core granules. This case stained positively for NSE (X28,OOO).

and six are dead (three with positive markers). Among the four patients with tumors less than 8 cm, one is alive (negative for markers) and three are dead (one with pos- itive markers).

as NSE".18-'0 and Leu 7.6.2'.'2 Cavazzana et aL3 using ES tumor cell lines, showed that the neural features became more evident after inducing differentiation with cyclic adenosine monophosphate (CAMP) and phorbol esters. Neural features have also been described in extraosse- ous ES.23.24

Initially, ES with neural features detected by ultra- structure was regarded as a variant of ES.I7 Subsequently, with the use of immunohistochemical stains for neural markers such as NSE and Leu 7, these tumors were named

Discussion

The presence of neural features in small round cell sar- comas of bone previously diagnosed as ES has been doc- umented by electron microscopic e ~ a m i n a t i o n ~ . ' ~ - ' ~ and immunostaining with antibodies to such neural antigens

TABLE 2. Site of the Tumor at the Time of Diagnosis Correlated With Survival and Neuron-Specific Enolase/Leu 7 Staining

Site No. of cases L/ M RT Alive Dead

Skull Lower extremities Pelvis Vertebrae Ribs Upper extremities Scapula

Total

3 - 2 L 14 12 L/2 M 6 5 L/I M I I L 9 5 L/4 M 4 3 L/1 M 4 3L/1 M

40 31 L/9 M

2 CR 13 CR/I NR

5 CR/I NR CR

6 CR/3 N R 4 CR 3 CR/1 PR

34 CR/I PR 5 NR

L: localized; M: metastatic: RT: response to therapy; CR: complete * ( ): Number of cases positive for NSE/Leu 7 (immunoperoxidase). remission: PR: partial remission: NR: no remission.

No. 6 NEURAL MARKERS IN EWING'S SARCOMA * Pinto et al. 1271

P-.0495

neuroectodermal tumors of bone7,18,19,25-27 and were in- cluded among the PNET2' Clinically and radiologically these tumors are indistinguishable from ES and are treated as such. The pathologic diagnosis of neuroectodermal tu- mors of bone is challenging. Currently, immunostaining with NSE and Leu 7 is the most useful technique for di- agnosis. The validity of the above markers has been con- firmed by the ultrastructural findings of dense core gran- ules and microtubules in dendritic processes from ES ce11s.7.18,25-27 A controversy has arisen over whether every small round cell sarcoma of bone which displays NSE or Leu 7 positivity should be considered a neuroectodermal tumor and therefore different from ES of mesenchymal nature, and whether ES with positive NSE or Leu 7 stain- ing should be treated differently.

The light microscopic appearance of ESB and neu- roectodermal tumors is similar. Rosettes with fibrillary cores and a lobular pattern have been described in both. 16,18,19,25,29 Th e presence of rosettes is typically focal and in our series three of the five cases with rosettes stained weakly with NSE and/or Leu 7.

The NSE staining in HWR has been reported as weakly positive to negati~e.~' Neuron-specific enolase, one of the isoenzymes of enolase, is a dimer of two gamma subunits that is present in neurons and in peripheral and central neuroendocrine cells.31 The nonneural tumors reported showing positivity with NSE32 did not present a differential diagnostic problem in our series of ESB. The usefulness of NSE in the diagnosis of small round cell sarcomas of childhood has been documented by Triche ef a1.,20 who tested a number of antisera to NSE, and by Tsokos et aI.,33 who tested a rabbit antibody against rat NSE. Both studies found that all neuroblastomas stained regardless of their degree of differentiation. Only one embryonal rhabdomyosarcoma stained among the other small round cell sarcomas of childhood. The diagnosis of rhabdomyo- sarcoma was excluded from our series because all tumors were negative for CKMM and desmin. A monoclonal an- tibody against NSE has been recently described and ap- pears to be more specific33; however, in a recent report of neuroectodermal tumors of bone no differences in the staining were reported between the use of monoclonal and polyclonal a n t i b ~ d i e s . ~ ~ The pattern of NSE staining in our cases was similar to that of previously reported cases of ESB and neuroectodermal t ~ m o r s . ~ , ~ , ~ ~ In our series the five cases regarded as atypical were positive and three of the five cases with rosettes were positive for NSE. A greater number of cases is necessary to make a definitive statement about the relationship between neural markers and histology.

Leu 7, an IgM monoclonal antibody described as a marker for large granular lymphocytes which possess nat- ural killer cell activity,34 has been found to recognize my- elin-associated glycoprotein in peripheral and central

1.0

0.8

0.6 0 .- L

k 2 0.4 n

0.2

Positive, Localized Negative, Metastatic Negative, Localized Positive, Metastatic

................,

- _____ .... ..............

15 17 0 5 10

Years

FIG. 7. Kaplan-Meier estimates of the probability of survival in patients with Ewing's sarcoma of bone according to NSE/Leu 7 immunostaining and extent of disease.

nervous system and in tumors originating in these loca- t i o n ~ . ~ ~ Recently Leu 7 positivity has been reported in prostate.36 Among small cell sarcomas Leu 7 stains neu- roblastoma, primitive neuroectodermal tumors and ES.6321,22*25926 In our series 12 cases were positive for Leu 7 and eight cases were positive for both NSE and Leu 7.

On eight cases reactive with anti-NSE, we performed immunostaining with SY38, a monoclonal antibody that recognizes synaptophysin, a membrane glycoprotein found on presynaptic vesicles which has been proposed as a novel marker of neuroendocrine cells and tu- m o r ~ . ~ ~ - ~ ~ The reactivity of the epitope recognized by SY38 is optimal in frozen tissue and less than optimal in conventionally fixed and paraffin-embedded Bouin's solution appears to be a better fixative than form- aldehyde for the preservation of the epitope recognized by SY38.38,39 Our lack of reactivity of SY38 with all but one of the cases tested may reflect the small amount of expression or a failure of our methodology. It is of im-

1.0

0.8 t '"n I L;-L

0.2 1 5 Negative , ----LA

1272 CANCER September 15 1989 Vol. 64

portance that the one case which stained with SY38 also showed the highest degree of NSE expression.

Dense core granules, dendritic processes, and micro- tubules have been described by ultrastructure in

and ES cell lines after inducing differentiation with CAMP.^ The interpretation of the dense core granules as true neurosecretory granules is difficult because they can be confused with primary lysosomes, as has been re- ported in non-Hodgkin's lymph~ma.~ ' Ultrastructural staining with the uranaffin reaction4' tends to support the view that in some ESB these are true neurosecretory gran- u l e ~ . ~ In diagnostic electron microscopic study the neu- rosecretory nature of the dense core granules is supported by staining with NSE in paraffin sections. These neural features are found preferentially when HWR are present for ultrastructural examination. In our series only one case with HWR was available for electron microscopic examination and showed typical dense core granules and dendritic processes as well as positivity with NSE. In five cases with atypical dense core granules by ultrastructure, the immunostaining with NSE or Leu 7 was negative re- flecting the possibility that these atypical dense core gran- ules may represent lysosomes.

H i s t ~ l o g i c ' ~ , ~ ~ and n o n h i s t ~ l o g i c ' ~ , ~ ~ parameters have been evaluated in the prognosis of ESB; no information is available, however, about the clinical relevance of NSE and/or Leu 7 staining. A predominant filigree att tern'^.^^ and intramyofiber muscle invasion4' have been reported to be associated with poor outcome. In our series four cases showed predominant filigree pattern; two patients are alive and two are dead. Five patients had tumors with intramyofiber muscle invasion, and all are dead. The presence of metastasis at diagnosis, with few exception^,'^ has been reported to be a poor prognostic indicator. In our series seven of the nine patients with metastasis at diagnosis achieved CR but only three are alive at the time of this report. The size of the primary tumor, when greater than 8 cm, is considered a poor prognostic ind i~a t0r . l~ In our series fifteen cases (ten localized and five with metas- tasis) had tumors greater than 8 cm. Due to this small number of cases, no correlation between size, staining and prognosis could be found.

Our results in this relatively small series show that ESB commonly expresses NSE and/or Leu 7 (58% of the cases in our series). This finding strengthens the biological and clinical similarities between ESB and neuroectodermal tumors of bone. The presence of NSE and/or Leu 7 stain- ing was of no prognostic significance in the overall survival of these patients. Nevertheless, in our studies, all six pa- tients with positive NSE and/or Leu 7 staining who failed induction therapy died of disease within 2 years. However, in patients with localized tumors that respond to induction therapy, the presence of NSE and/or Leu 7 staining may be a favorable prognostic indicator.

ESB7,I7.18.'5

REFERENCES

I . Ewing J. Diffuse endothelioma of bone. Proc N Y Pafhol Soc 1921; 2 1 : 17-24,

2. Moll R, Lee I , Could VE, Berndt R, Roessner A, Franke W. Im- munocytochemical analysis of Ewing's tumors. Am J Pathol 1987; 127:

3. Cavazzana AD. Miser JS, Jefferson J, Triche TJ. Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pafhol

4. Lipinsky M. Hirsch MR, Deagostini-Bazin H, Yamada D, Turz T, Gondis C. Characterization of neural cell adhesion molecules (NCAM) expressed by Ewing's and neuroblastoma cell lines. Int J Cancer 1987; 80:81-86.

5. Lipinsky M. Braham K, Philip I et al. Neuroectodermal-associated antigens on Ewing's Sarcoma cell lines. Cancer 1987; 47:183-187.

6. Kawaguchi K. Koike M. Neuron-specific enolase and Leu 7 im- munoreactive small round cell neoplasm: The relationship to Ewing's Sarcoma in bone and soft tissue. A m J Clin Pathol 1986: 86:79-83.

7. Llombart-Bosch A, Lacombe M, Contesso G, Peydro-Olaya A. Small round cell Sarcomas of bone mimicking atypical Eying's sarcoma with neuroectodermal features: An analysis of five cases with immu- nohistochemical and electron microscopic support. Cancer 1987; 60: 1570-1582.

8. Whang-Peng J. Triche TJ, Knutsen T , Miser J, Douglass EC, Israel MA. Chromosomal translocations in neuroepithelioma. N Engl J Med 1984; 311584-585.

9. Aurias A. Rimbaut C, Buffe D, Dubousset J, Mazabraud A. Chro- mosomal translocations in Ewing's sarcoma. N Engl J Med 1983; 309:

10. Turc-Carel C. Philip T, Lenoir GM, Berger M. Chromosomal translocations in Ewing's Sarcoma. N Eng/ J Med 1983; 309:497-498.

1 1. Thiele C, McKeon C, Triche TJ, Ross RA, Reynolds P, Israel M. Differential proto-oncogene expression characterizes histopathologically indistinguishable tumors of the peripheral nervous system. J Clin Invest 1987; 80304-811.

12. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochern Cytochem 1981; 295777580,

13. Wordinger RJ, Miller GW. Nicodemus DS. Manual of Immu- noperoxidase Techniques, ed. 2. Chicago: American Society of Clinical Pathologists. 1987; 49-50.

14. Hayes FA. Thompson EI, Rao B, Kun L, Parham D. Hustu HO. Metastatic Ewing's sarcoma. J CIin Oncol 1987; 5: 1199-1204.

15. Pet0 R. Pet0 J. Asymptotically efficient rank invariant test pro- cedures (with discussion). J R Stat Soc [A] 1972; 135:185-206.

16. Kissane J, Askin F, Foulkes M, Stratton LB, Shirley SR. Ewing's sarcoma of bone: Clinicopathologic aspects of 303 cases from the Inter- group Ewing's Sarcoma Study. Hurn Pathol 1983; 14:773-779.

17. Schmidt D. Mackay B. Ayala AG. Ewing's sarcoma with neuro- blastoma-like features. Liltrasfrtrcf Patho/ 1982; 3: 143-1 5 1.

18. Jaffe R, Santamaria M. Yunis EJ et a/. The neuroectodermal tumor of bone. A m J Surg Pathol 1984; 8:885-898.

19. Yunis E. Ewing's Sarcoma and related small round cell neoplasms in children. A m J Sirrg Palhol 1986; (Suppl) 1054-62.

20. Triche TJ, Tsokos M, Linnoila RI, Marangos PJ, Chandra R. NSE in neuroblastoma and other round cell tumors of childhood. In: Evans AE, D'Angio GJ, Seeger DC, eds. Advances in Neuroblastoma Research. New York: Alan R Liss, 1985; 295-317.

2 I . Michels S. Swanson PE, Robb JA, Wick MR. Leu 7 in small cell neoplasms an immunohistochemical study with ultrastructural corre- lations. Cancer 1987; 60:2958-2964.

22. Donner L, Triche TJ, Israel MA, Seeger RC, Reynolds CP. A panel of monoclonal antibodies which discriminate neuroblastoma from Ewing's sarcoma, rhabdomyosarcoma, neuroepithelioma and hemato- poietic malignancies. In: Evans AE, D'Angio EJ, Seeger RC, eds. Ad- vances in Neuroblastoma Research. New York: Alan R Liss, 1985; 347- 360.

23. Shimada H. Newton W. Soule E, Qualman SJ. Aoyama C, Maurer HM. Pathologic features of features of extraosseous Ewing's sarcoma: A

288-304.

1987; 127507-5 18.

496-497.

No. 6 NEURAL MARKERS IN EWING’S SARCOMA . Pinto et al. 1273

report from the Intergroup Rhabdomyosarcoma Study. Hum Pathol

24. Mierau GW. Extraskeletal Ewing’s sarcoma (peripheral neuroep- ithelioma). Ultrastruct Pathol 1985; 9:91-98.

25. Llombart-Bosch A, Lacombe MJ, Peydro-Olaya A, Perez-Bacete M, Contesso G. Malignant peripheral neuroectodermal tumors of bone other than Askin’s neoplasm: Characterization of 14 new cases with immunohistochemistry and electron microscopy. Virchows Arch [A]

26. Jurgens H, Bier V, Harms D et a/. Malignant peripheral neu- roectodermal tumors. Cancer 1988; 61:349-357.

27. Schmidt D, Harms D, Burdach S. Malignant peripheral neuroec- todermal tumors of childhood and adolescence. Virchows Arch [A] 1985;

28. Dehner LP. Peripheral and central primitive neuroectodermal tu- mors: A nosologic concept seeking a consensus. Arch Pathol Lab Med

29. Llombart-Bosch A, Contesso G, Henry-Amar M, Lacombe M. Histopathological predictive factors in Ewing’s sarcoma of bone and clinicopathological correlations. Virchows Arch [A] 1986; 49~627-640.

30. Hashimoto H, Enjoji M, Nakajima T, Kiryu H, Daimaru Y . Ma- lignant neuroepithelioma (peripheral neuroblastoma). Am J Surg Pathol

31. Marangos PJ, Polak JM, Pearse AGE. Neuron specific enolase: A probe for neurons and neuroendocrine cells. Trends Neurosci 1982; 5:193.

32. Vinores SA, Bonnin JM, Rubinstein J et al. Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other tissues. Arch Pathol Lab Med 1984; 108536-540.

33. Thomas P, Battifora H, Manderino G , Patrick J. A monoclonal

1988; 19:442-453.

1988; 412~421-430.

406~35 1-365.

1986; 110~997-1005.

1983; 7~309-318.

antibody against neuron-specific enolase. Am J Clin Pathol 1987; 88:

34. Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK 1). J Immzrnol198 1 ;

35. McGany RC, Helfand SL, Quarels RH, Roder JC. Recognition of myelin associated glycoprotein by the monoclonal antibody HNK 1. Nature 1983; 306:376-378.

36. Rusthoven JJ, Robinson JB, Kolin A, Pinkerton PH. The natural killer cell associated HNK I (Leu 7) reacts with hypertrophic and ma- lignant prostatic epithelium. Cancer 1985; 56:289-293.

37. Wiedenmann B, Franke WW, Kuhn C et al. Synaptophysin: A marker protein for neuroendocrine cells and neoplasms. Proc Nut1 Acad Sci USA 1986; 83:3500-3504.

38. Could VE. Weidenmann B, Lee I et a/. Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry. Am JPathol 1987; 126:243-257.

39. Chejfec G, Falkmer S, Grimelius L et al. Synaptophysin: A new marker for pancreatic endocrine tumors. A m J Surg Pathol 1987; 1 1 :

40. Herrera GA, Crofts JL, Roberts CR. Neurosecretory granule-like structures in lymphomas. Hum Pathol 1980; 11:449-457.

4 1 . Richards JG, Da Prada M. Uranaffin reaction: A new cytochemical technique for the localization of adenine nucleotides in organelles storing biogenic amines. J Histochem Cytochem 1977; 25: 130.

42. Stratton B, Askin F, Kissane J. lntramyofiber skeletal muscle in- vasion in Ewing’s sarcoma of bone: Clinicopathologic observations from the Intergroup Ewing’s Sarcoma Study. Am J Pediatr Hematol Oncol

146-1 52.

127~1024-1029.

241-247.

1982; 4:231-135.