ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 25, No. 1 Copyright © 1995, Institute for Clinical Science, Inc.

Immunohistology, Cytogenetics, and Molecular Studies of Small Round Cell Tumors of Childhood

A Review*

JEFFREY L. W INTERS, M .D.,t JOHN D. GEIL,

and WILLIAM N. O’CONNOR, M .D.t

fDepartment o f Pathology and Laboratory Medicine tD epartm ent o f Pediatrics,

College of Medicine, University o f Kentucky, Lexington, KY 40536

ABSTRACT

Malignancies of childhood include a well-defined spectrum of hema- tolymphoid, organ specific (adrenal, kidney, liver), soft tissue, bone, and nervous system (central and peripheral) neoplasms with variable biology. Small round cell neoplasms, a subset of childhood malignancies, are his­tologically similar bu t differ markedly in their histogenesis, therapy, and prognosis. Traditionally, clinical information and light microscopy, with the aid of histochemistry and ultrastructural evaluation, establish a diag­nosis or at least narrow the differential diagnosis. Additionally, imm uno­histology, cytogenetics, and m olecular studies have become im portant in diagnosis and in defining phenotype/genotype, patient treatm ent modali­ties, and prognosis in specific cases. The 11;22 chromosomal translocation typifies Ew ing’s sarcoma, prim itive neuroectodermal tumor, and Askin’s tumor, as does the resultant chim eric transcript, while expression and amplification of N-myc oncogene are predictive of the prognosis in neuro­blastoma. Furtherm ore, studies of genes and gene products are elucidating m echanisms of oncogenesis and tumor progression.

Introduction

Traditional and tim e-honored clinical and laboratory studies have shown that tumors occurring in childhood have a dif­ferent histology and biological behavior than those commonly seen in adults. The focus of this article is the expanding role of im m unohistology, cytogenetics, and m olecular studies of childhood malignan­cies, especially the small round cell neo­

plasm s. M ajor tum ors are lis ted w ith p e rtin e n t dem ograph ics, usual c lin i­cal behavior, and re lev an t com m ents on histogenesis.

Tumor Types

N EUROBLASTOMA

This is the most common solid tumor of childhood and infancy excluding central

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STUDIES O F SMALL ROUND CELL TUMORS O F CH ILD H O O D 67

nervous system (CNS) tum ors. I t is derived from neural crest and most com­monly occurs in the adrenal m edulla and thorax. The tum or is frequently m eta­static at the tim e of initial diagnosis . 1

E w i n g ’s S a r c o m a (E S )

This typically arises in bone, but may also develop in soft tissue (extraosseous E w in g ’s sarcom a). E w in g ’s sarcom a tends to develop distant metastases. In vitro exposure of ES cells to growth fac­tors results in the expression of neural enzymes and proteins, which suggests a neural h istogenesis . 2 E w ing’s sarcoma appears to be closely related to primitive neuroectoderm al tum ors .3 ,4

P r i m i t i v e N e u r o e c t o d e r m a l T u m o r ( P N E T )

This is predom inantly an extraskeletal tumor that is clinically more aggressive than E w in g ’s sarcom a . 5 T he PN ETs show antigens and cytogenetic abnor­m alities3,4 identical to those found in ES and therefore these tumors appear to be closely related.

A s k i n ’s T u m o r (A T )

This is a rare tum or of the chest wall, possib ly arising w ith in costal nerves. There is a female predom inance. Askin’s tumor is locally aggressive, bu t does not metastasize. Typically, AT has a poorer prognosis than ES 5 ,6 and appears to be related to PN ET and ES as they share a common cytogenetic abnorm ality .6

M e d u l l o b l a s t o m a

This is the most common neuroecto­dermal tumor of the CNS. M edulloblas­toma most often originates in the midline of the cerebellum and has a propensity to invade the ventricular system with cere­brospinal fluid (CSF) dissem ination .7

R e t i n o b l a s t o m a

This is the m ost common intraocular neoplasm occurring during the first 15 years of life. Six percent of cases repre­sen t autosom al dom inant inheritance. Local spread and dissem ination via CSF are seen, bu t distant metastases are rare .7

R h a b d o m y o s a r c o m a

This most commonly develops in the soft tissue of the head, neck, and geni­tourinary tract. It has a slight male pre­dominance and two peaks of occurrence: one in in fa n c y an d th e o th e r d u r ­ing adolescence .8

H e p a t o b l a s t o m a

This most often presents in children less than tw o years o f age and p re ­dominantly involves the right lobe of the liver. Anaplastic tumors must be differen­tia ted from m etasta tic neuroblastom a and rhabdomyosarcoma .8

W i l m ’s T u m o r (n e p h r o b l a s t o m a )

This is characteristically triphasic, but can present as a monophasic tumor. It is thought to arise from residual blastemal rests w ithin the periphery of the kidney . 9

M a l i g n a n t R h a b d o i d T u m o r (M R T )

This is a rare but highly lethal neo­plasm of early life with a mortality rate exceeding 80 percent . 10 The MRT pre­dominantly arises w ithin the kidneys and is a s so c ia te d w ith c o n c u rre n t CNS tum ors (i.e., m edulloblastom a). W hile MRT is thought to be of neural or neuro­endocrine origin , 10 this tumor has also b een rep o rted a ris ing in the liv e r , 11

uterus , 12 soft tissues , 13 and brain . 10

68 WINTERS, GEIL, AND O’CONNOR

C l e a r C e l l S a r c o m a o f Ki d n e y (C C S K )

This is an uncommon tum or of kidney, p resen ting predom inan tly in ch ildren less than six years of age. It has a pro­clivity for bone m etastases, especially the skull. 14

O s t e o s a r c o m a

This is the m ost common m alignant bone forming tum or in children and ado­lescents. It metastasizes early and has a poor p rognosis . 15 A small cell variant exists which has an even worse prognosis and m ust be differentiated from ES . 16,17

E p i t h e l i o i d Sa r c o m a

This is a rare tumor that has a slight m ale p red o m in an ce and occurs m ost comm only on the fingers, hands, and fo rearm s o f a d o le s c e n ts an d young adults. M ultiple recurrences and metas­tases are common. The tum or is often confused w ith granulomatous infections, squamous cell carcinoma, or melanoma . 18

H e m a t o l y m p h o i d N e o p l a s m s

T h e tw o m o s t c o m m o n tu m o rs in c lu d ed in the d iffe ren tia l of small round cell tum ors are Burkitt’s (small noncleaved) and lymphoblastic lympho- mas/leukemias. Burkitt’s lymphoma is a m alig n an cy o f B -cell o rig in . In the United States, it most commonly presents as an abdom inal mass. Lym phoblastic lymphoma is a high-grade T-cell malig­nancy, which often presents with a m edi­astinal m ass . 19

Histology

W hile the focus of this article is the expanding use of immunohistology, cyto­genetics, and m olecular studies, it should be stated that routine light microscopy

with or without histocytochemistry and electron microscopy are the mainstays of d iagnosis. L igh t and e lec tron m icro­scopic features of the various tum ors listed previously are described in stan­dard texts th a t are w idely availab le . W hile outside the scope of this paper, the subset of small round cell neoplasms con­sisting of neuroblastoma, ES, PNET, and AT show considerably overlap betw een histologic and ultrastructural features. This subset of small round cell n eo ­plasms of childhood can, how ever, be segregated using the criteria lis ted in table I.

Immunohistology

The reactivity of the tumors to a variety of antibodies is shown in tables II and III. The reactivity is variable, dependen t not only upon the preparation of the specim en {e.g., alcohol versus formalin fixation versus frozen section) and the antibody used, but also the degree of dif­ferentiation of the tumor. Examples of the latter include the expression of myo­globin in rhabdomyosarcoma2 0 and the expression of various antigens in medul- loblastom a. In m edu llob lastom a, th e antigens expressed are dependent upon the line of differentiation of the tumor as seen in desmoplastic versus the classic varieties .21 This is also the cases in hepa­toblastoma where the various tumor com­ponents will have different reactivity . 22

As show n in tab le III , the degree of immunologic characterization of the indi­vidual small round cell tumors reported w ithin the literature is quite variable. As a resu lt, the com parison o f d iffe ren t tumors is very difficult in some cases. T h is re p re se n ts an a rea in n e e d o f future research.

Certain single antibodies may be use­ful in narrow ing the differential or in m aking th e d iagnosis. For exam ple, desmin is useful for separating rhabdo­myosarcoma from Ewing’s sarcoma, neu­roblastom a, and hem atolym phoid neo­

STUDIES O F SMALL ROUND C ELL TUMORS O F C H ILD H O O D 6 9

TABLE I

Morphology of Selected Small Round Cell Neoplasms ab

NeuroblastomaEwing’s

Sarcoma

PrimitiveNeuroectodermal

TumorAskin’sTumor

Nuclear detail Roundhyperchromatic speckled nuclei

Regular with finely stippled chromatin

Densehyperchromaticchromatin

Round with finechromatin

Mitoses Few to many0 Variable High Occasional

Glycogen Not as common as in ES

Prominent Not as common as in ES

Not present

Rosette formation Well-formedrosettes

Pseudorosettes More well-formed true rosettes

Pseudorosettes

Overall pattern Nests separated by fibrous septa

Cells arranged in sheets and lobules

Cells arranged in sheets and lobules

Radially oriented peripheral cells

Ultrastructuralfeatures

Prominentcellularprocesses and dense core granules

Few organelles, glycogen pools

Cellular processes and occasional dense core granules

Few organelles, neurosecretory granules and and microtubules present

Other Increasedcatecholamines

Arise from large nerve trunks

Normalcatecholamines

aDehner LP. Pediatric Surgical Pathology. Baltimore, Williams & Wilkins, 1987. bEnzinger FM, Weiss SW. Soft Tissue Tumors. St. Louis, The C.V. Mosby Company, 1988, pp.

936-45.cCan show considerable mitoses in undifferentiated areas with associated karyorrhectic debris.

plasms. Also, HBA-71 has been shown to be expressed on ES and PN ET 3,23 as well as ependymomas, islet cell tumors, glio­blastomas, and rhabdomyosarcomas .24 It is not clear in the literature w hether or no t expression in A skin’s Tum or has been evaluated. Another useful single antibody is leukocyte common antigen (LCA) w hich can be used to separate hematolym phoid malignancies from the rem ainder of small round cell tumors.

W hile these examples show that single antibodies are useful, there is no anti­body specific for a single tumor type. In

addition, there is overlap of m esenchy­mal, epithelial, and neural markers in a variety of tumors as shown by comparing expression of neuron specific enolase (NSE) (see table III). Because of this, panels of antibodies are m ore helpful. T here are, how ever, stain ing patterns that are helpful for individual tum ors such as osteocalcin positivity in osteo­sarcomas15 and the staining of epithelioid sarcomas by CD34, alpha 1 antitrypsin, and alpha 1 antichymotrypsin .34

Immunohistology has also been shown to be prognostically significant in neuro-

TABLE II

70 W INTERS, GEIL, AND O ’CONNOR

Immunohistology of Selected Related Small Round Cell Tumors of Childhood*

NeuroblastomaEwing’s

Sarcoma

PrimitiveNeuroectodermal

TumorsAskin’sTumor

Neuron specific enolase + a ± b 4- a,c + b.dNeurofilament + 0 — C + ° _e

S-100 + c _ c + cHNK-1 _ c __ c + °Ganglioside GD2 + * _ f

HBA-71 - c,g + g.h + g.hCytokeratin _ i — i ± 0 + eVimentin j + i + c + eActin _ c — c ± 0Desmin _ i — C.i ± 0 _e

Leukocyte common antigen _ i _ j _ c

+ = Reactive - = Non-reactive ± = Variable reactivity‘ Medulloblastoma (a central nervous system tumor) is generally positive for S -1 00, ganglioside

GD2, and alpha fetoprotein (Cruz-Sanchez FF, Rossi ML, Hughes JT, Esiri MM, Coakham HB. Acta Neuropathol 1989;79:205-10).

a Parham DM, Webber B, Holt H, Williams WK, Maurer H. Cancer 1991 ;67:3072-80. b Triche TJ, Askin FB. Hum Pathol 1983;14:569-94. c Dehner LP. Am J Surg Path 1993;17:1-13.d Seemayer TA, Vekemour M, de Chadarevian JP. Virchows Arch 1985;408:289-96.9 Noguera R, Navarro S, Triche TJ. Cancer Genet Cytogenet 1990;45:121-4.1 Cheung NV, Saarinen UM, Neely JE, Landmeier B, Donovan D, Coccia PF. Cancer Res 1985;45:

2642-9.9 Ambros IM, Ambros PF, Strehl S, Kovar H, Gadner H, Salzer-Kutschik M. Cancer 1991 ;67:

1886-93.h Fellinger EF, Garin-Chesa P, Glasser DB, Huvos AG, Rettig WJ. Am J Surg Path 1992;16:746-55. ' Pilkinton GR, Pallesen G. Histopathology 1989;14:347-357. i Oppedal BR, Storm-Mathisen I, Lie SO, Brandtzaeg P. Cancer 1988;62:772-80.

blastoma. An increase in expression of LCA is associated with a poor prognosis. T his rep re sen ts increased leukocytic infiltration of the tumor and is associated with increased karyorrhexis. Therefore, increased expression w ould be associ­ated histologically with an elevated mito- sis-karyorrhexis index (MKI) and may not be an independent indicator of the prog­nosis. The MKI is a com ponent of the Shimada classification, a prognostic indi­cator that also includes the am ount of stroma, degree of differentiation, and the patient’s age .25 An increased expression of S-100, on the other hand, would be

an ind ica to r of good prognosis since it w ould im ply a m ore d iffe ren tia ted tumor stroma.

The studies summarized in tables II and I II used form alin fixed, paraffin- em bedded 3 ’10’15’2 0 ’2 1 ’2 2 ’3 0 ’3 1 ’3 3 ’34 and fro­zen tissue .23 ,2 7 ,2 9 In some instances, both frozen and form alin fixed tissue w ere used 2 8 ’32 w hile in others, the type of specimen was not specified . 1,5 The opti­mal processing depends upon the anti­body used for staining. In addition, some a n tib o d ie s r e q u ir e th e p ro te o ly tic enzyme treatm ent of paraffin em bedded tissue. Freezing, most often, is less likely

TABLE II!

STUDIES O F SMALL ROUND CELL TUMORS O F C H ILD H O O D 7 1

Immunohistology of Small Round Cell Tumors of Childhood “Exclusive of Neuroblastoma and Related Tumors"*

Hemato­ Rhabdo- Malignant Clear Cell Hapto-lymphoid myo- Rhabdoid Epithelioid Wilm’s Sarcoma blas-

Neoplasms Sarcoma Tumor Sarcoma Tumor of Kidney toma

Neuron specific - a + a — Nb

1 o >

+

1 _ d

enolaseNeurofilament - a ± a - N b _ c

S - 1 0 0 - a _ a - N b" - e - d

Ganglioside GD2 - f ± f _ f

Cytokeratin - a — a,g,h + b + « ± V ' _ d + V iEpithelial membrane ± b + k _ d

antigenCarcinoembryonic _ b ¿ 6 _ d

antigenAlpha fetoprotein _ b + V iVimentin ± a + a,i + d + 6,1 _ i + dDesmin - a + a,g,i - N d _ k ± ¡ _ d

Myoglobin + 9 - N d _ dDystrophin - m + m _m

Leukocyte common + ' — a - N d - 'antigen

a Dehner, LP. Am J Surg Pathol 1993;17:1-13.b Weeks, DA, Beckwith JB, Mierav GW, Luckey DW. Am J Surg Pathol 1989;13:439-58.0 Parham DM, Webber B, Holt H, Williams WK, Maurer H. Cancer 1991 ¡67:3072-80. d Oda H, Shiga J, Machinami R. Cancer 1993;71:2286-91.e Daimarv Y, Hashimoto H, Tsuneyoshi M, Enjoji M. Cancer 1987;59:134-41. f Cheung NV, Saarinen UM, Neely JE, Landmeier B, Donovan D, Coccia PF. Cancer Res 1985;

45:2642-9.9 Kodet R. Pathol Res Pract 1989;185:207-13.h Oppedal BR, Storm-Mathisen I, Lie SO, Brandtzaeg P. Cancer 1988;62:772-80.' Pilkinton GR, Pallesen G. Histopathology 1989;14:347-57.1 Abenoza P, Manivel JC, Wick MR, Hagen K, Dehner LP. Hum Pathol 1987;18:1025-35. k Arber DA, Kandalaft PL, Mehta P, Barrifora H. Am J Surg Pathol 1993;17:302-7.1 Mukai M, Torikata C, Iri H, Hanaoka H, Kawai T, Yakumaru K, Shimoda T, Mikatol A, Kageyama K.

Am J Pathol 1985;119:44-56.m Pinto A, Paslawski D, Sarnat HB, Parham DM. Mod Pathol 1993;6:679-84.N = Non-specific staining of inclusions.V = Varies among tumor components.

than formalin fixation, with or without enzyme treatm ent, to alter antigen struc­ture. In the end, the recommendations of the m an u fac tu rer of th e an tibody shou ld be fo llow ed regard ing sp ec i­m en preparation.

Cytogenetics

In some hematolymphoid neoplasms, cy togenetic abnorm alities, espec ia lly translocations, routinely aid in or estab­lish a diagnosis. With solid tumors, how-

72 W INTERS, GEIL, AND O ’CONNOR

TABLE IV TABLE V

Diagnostically Useful Cytogenetic Abnormalities

Ewing’s sarcoma t(11 ;22)aAskin’s tumor t(11 ;22)bcPrimitive neuro- t(11 ;22)a

ectodermal tumor Alveolar t(2;13)a

rhabdomyosarcom a Embryonal +2q, +20 a

rhybdomyosarcoma Neuroblastoma del 1p, double minutes dWilm’s tumor dup 1q, del 11p 8Retinoblastoma d e l1 3 q 1 4 f'9Hepatoblastoma +2, + 2 0 hMeduiioblastoma Isochromosome 17q 'Burkitt's lymphoma t(8;14) i

a Fletcher JA. Adv Pathol Lab Med 1991 ;4: 235-46.

b SeemayerTA, Vekemour M, de Chadarevian JP. Virchows Arch 1985;408:289-96.

0 Fujii T, Hongo T, Nakagawa Y, Nasuda K, Mizuno 0 , Igarashi Y, Naito Y, Maeda M. Cancer 1989;64:43-51.

d Fletcher JA, Kozakewich HP, Hoffer FA, Lage JM, Weidner N, Tepper R, Pinkus GS, Morton CC, Corson JM. New Engl J Med 1991 ;324:436-42.

8 McGavran L, Waldstein G, Beckwith B, Collins J, Giifiiian S. Am J. Hum Genet 1985;37:A32.

•Becker LE, Hinton D. Hum Pathol 1983;14: 538-49.

9 Yokoyama T, Tsukahara T, Nakagawa C, Kikuchi T, Minoda K, Shimatake H. Cancer 1989; 63:2134-8.

h Fletcher JA, Kozakewich HP, PavelkaK, Grief HE, Shamberger RC, Korf B, Morton CC. Genes Chromosomes Cancer 1991 ;3:37-43.

' Cogen PH, Daneshvar L, Metzger AK, Edwards MS. Genomics 1990;8:279-85.

1 Chaganti RS, Klein EA. Molecular Genetics in Cancer Diagnosis. Crossman J., ed. New York, Elsevier, 1990;73-104.

ever, these stud ies have not been as widely used for diagnosis as they have for prognosis. In table III is shown a current list of the m ost common diagnostically useful chromosomal abnormalities for a selected group of childhood tumors. It should be noted, how ever, that other

Cytogenetic Abnormalities Indicating Poor Prognosis

Neuroblastoma del(1 )(p31 —»32), HSR, double minutes, diploldy a Complex karyotype b oWilm’s tumor

(nephroblastoma)Hepatoblastoma AneuploidydRhabdomyosarcoma Diploidy 8 Meduiioblastoma DiploidybOsteosarcoma Absence of a near diploid

cell linefChildhood acute Diploidy 9

lymphoblastic leukemia

a Fletcher JA, Kozakewich HP, Hoffer FA, Lage JM, Weidner J, Tepper R, Pinkus GS, Morton CC, Corson JM. New Engl J Med 1991 ¡324:436-42.

b McGavran L, Waldstein G, Beckwith B, Collins J, Gilfillan S. Am J Hum Genet 1985;37:A32.

0 Molenaar WM, Dam-Meiring A, Kamps WA, Cornelisse CJ. Hum Pathol 1988;19:573-9.

d Hata Y, Ishizu H, Ohmori K, Hamada H, Sasaki F, Uchino J, et al. Cancer 1991 ¡68: 2566-70.

8 Shapiro DN, Parham DM, Douglass EC, Webber BL, Newton WA, Maurer HM, Look TA. Proc. Ann Meet Am Soc Clin Oncol 1990;9:290.

* LookTA, Douglass EC, Meyer WH. New Engl J Med 1988;318:1567-72.

9 Knowles DM. Neoplastic Hematopathology. Baltimore, Williams & Wilkins, 1992.

abnormalities have been reported less fre­quently. With biological tumor progres­sion, additional changes in karyotype may develop. Additionally, some cytogenetic abnormalities suggest a common histoge- netic rela tionsh ip be tw een ch ildhood tumor types. This is most apparent among E w ing’s sarcoma, PN ET, and A skin’s tumor in which a common translocation, t(ll;22) is frequently found. This charac­te r is t ic tra n s lo c a tio n has also b e e n reported in some cases of small cell osteo­sarcoma36 and esthesioneuroblastoma .37

In some childhood tumors, cytogenetic abnormalities have been found to be of

STUDIES O F SMALL ROUND CELL TUMORS O F CH ILD H O O D 73

prognostic significance, a current listing is summ arized in table IV. The presence of specific cytogenetic abnormalities in neuroblastom a38 and W ilm’s tumor39 has been associated w ith m ore aggressive behavior and a poorer response to che­m otherapy. I t should be noted that, in general, d ip lo idy is a poor prognostic finding in pediatric tumors, in contrast to adult tumors w here aneuploidy is a poor prognostic finding.

Classical m ethods of karyotype analy­sis involve the culture of tum or cells, with production of m etaphase spreads. Such m ethods require fresh tumor, not frozen or fixed/paraffin-embedded archi­val tissue. Small biopsies or non viable tum or tis su e sp ec im en s, even w hen freshly obtained, may also result in fail­ure to culture adequate num bers of cells for analysis. Recent use of fluorescent in situ hybridization (FISH) using deoxyri­b o n u c le ic a c id (D N A ) p ro b e s has a llow ed the iden tifica tion of specific cytogenetic abnormalities in fixed, paraf­fin em bedded tissue .4 0 The FISH tech­nique should be of benefit as it becomes more w idespread and as probes become available. Ploidy has traditionally been d e te rm in e d by m ea su rem e n t o f the uptake of radioactive nucleotides. More recently, however, flow cytometric anal­ysis using fluorescent dyes and image analysis has been used to study ploidy and its use is becoming more common.

M olecular Studies

M olecular studies involve the use of various techniques, such as fluorescence in situ hybridization (FISH), im m uno­p réc ip ita tio n , N orthern b lo t analysis, Southern blot analysis, and polymerase chain reaction (PCR) to determ ine the presence and level of oncogene DNA, mRNA, or protein product. W hile this is a rapidly expanding field of research, rou tine applications for diagnosis and m anagem en t o f ch ildhood tum ors as yet lim ited.

As with cytogenetic abnormalities, cer­tain patterns of tum or expression are potentially useful for diagnosis or prog­nosis. Until recently , it was generally accepted that expression and/or amplifi­cation of N-myc was diagnostically spe­cific for the grouping of neuroectoderm d e r iv e d tu m o rs . 5 1 H o w ev er, N -m yc amplification has been reported in some rh a b d o m y o s a rc o m a s , 5 2 a n d N -m yc expression has been found in cases of W ilm ’s tum or and h ep a to b las to m a . 5 3

Currently, m olecular studies are used routinely only for predicting prognosis in patients with neuroblastoma.

W ith neuroblastom a, 40 p e rc e n t of tumors show amplification or overexpres­sion of the N-myc oncogene. This often correlates with the presence of homoge­neously staining regions (HSRs) or dou­b le m inu te chrom osom es on p a ra lle l cy to g e n e tic s tu d ie s . T h e d e g re e of N-myc amplification in prim ary neuro­blastoma tissue is correlated w ith prog­nosis, where increased levels of N-myc indicate poor prognosis. T raditionally, N-myc expression has been m easured using Southern blot analysis, bu t FISH has also recently proved useful. It repre­sents an alternative and more rapid tech­nique that can be applied in routine h is­tologic secretions .54

The Trk proto-oncogene encodes a trans membrane tyrosine kinase, a por­tion of the neural growth factor (NGF) receptor .55 In neuroblastoma, high levels of Trk mRNA and protein expression are found in stage I, II, and IVS tum ors, while low levels are present in stage III and IV tum ors . 5 6 ,5 7 The T rk p ro te in expression and the level of mRNA in the m ajo rity o f s tu d ie d n eu ro b la s to m as inversely correlate with cytogenetic dou­ble m inute status and directly correlate with the histologic degree of differentia­tion . 55 Corroborating this finding in neu­roblastoma is the fact that N-myc am pli­fication is absent w hen there is elevation of Trk mRNA . 56 Because of these find­

74 W INTERS, GEIL, AND O’CONNOR

ings, it is postulated that increased Trk expression makes neuroblastom a more sensitive to the presence of N G F . 57 This may allow tumors presenting at stages associated w ith a better prognosis (I, II, or IVS), to differentiate or regress spon­taneously . 57 Furtherm ore, high levels of Trk expression or mRNA also appear to indicate a favorable prognosis in tumors without N-myc amplification . 57 Finally, high expression of Trk and N-myc (with­out amplification) is also associated with a favorable outcom e . 55

B esides th e re la tio n sh ip b e tw e e n N-myc and Trk, molecular studies of neu­roblastoma involving other cell surface molecules, enzyme metabolism, and pro­tein expression have shown interesting relationships to N-myc. F indings from these studies include downregulation of MHC class I antigens ,58 ,5 9 downregula­tion of neural cell adhesion m olecules .60

an inverse co rre la tion w ith ca techo l­amine m etabolism ,61 an inverse relation­ship in the expression of neural growth factor receptor ,62 and in inverse relation­ship with the expression of the m ultidrug resis tance gen e 6 3 in the p resen ce of N-myc amplification. In summary, such studies may in the future explain specific biologic behavior of these tumors.

In addition to its occurrence in neuro­blastoma, expression of N-myc may also predict poor prognosis in retinoblastoma. The histologic grade in retinoblastom a is a reported prognostic factor, with poorly d iffe ren tia ted tum ors having a w orse prognosis. Poorly differentiated retino­blastom a has been found to have e le ­va ted leve ls of N-myc gene p ro d u ct w ithou t ap p aren t gene am plification. These investigators showed absence of N-myc expression in ad jacent norm al

A n

eye structures.In ES, PN E T , and neurob lastom a,

m olecular stud ies have added further evidence to the theory that the first two are closely related. Studies summarized by Stephenson et al64 related an identical

pattern of oncogene expression in ES and PNET with expression of c-myc, c-src, c-myb, and c-mil/raf. Furtherm ore, ES and PNET variably express c-ets-1 and N-m yc, w h ile lack ing ex p ress io n of c-sis .64 C-sis is notably translocated from chromosome 22 to 11 in both ES and P N E T b u t is n e i th e r a l t e r e d n o r expressed in e ither .65 ,6 6 This pattern of oncogene expression contrasts with neu­roblastom a w hich lacks expression of c-myc and has more constant expression of c-ets-1 and N-myc .67

Differences in the expression of neural cell adhesion m olecule (NCAM) have also been shown betw een ES and neuro­blastoma .6 0 Neuroblastoma shows a long polysialic acid chain characteristic of fetal brain, w hile ES lacks this chain, and, in contrast, shows expression of a mature NCAM m olecule .60

Recently, a specific chimeric transcript has been reported in ES, PN ET, and AT .68 The transcript consists of the N-ter- minal portion of the EWS protein from chrom osom e 22 fused w ith the DNA binding domain of the FLI 1 transcrip­tion factor from chrom osom e 11. The resultant protein may alter transcription of yet unidentified genes. This chimeric protein corresponds with the characteris­tic 1 1 ; 2 2 translocation seen in these tumors and occurs in tumors displaying this cytogenetic abnormality. O f note, the chimeric protein was also seen in 6 of 8

tumors lacking the translocation and was no t seen in n e u ro b la s to m a or o th e r tum ors exam ined . 6 8 A nother chim eric protein consisting of EWS and ERG, a transcription factor homologous to FLI 1 and located on chromosome 2 1 , was seen in tumors with a t(21;22). Thus, Delattre e t a l6 8 p ro p o sed th e usage o f th ese chimeric transcripts for characterization of the E w ing’s fam ily of tum ors and argues that these tumors arise by a com­mon process.

A chimeric protein involving the PAX3 and FKHR gene products has also been

STUDIES O F SMALL ROUND CELL TUMORS O F C H ILD H O O D 75

identified in alveolar rhabdom yosarco­mas w ith t(2;13).69 This chim eric tran­script results from the fusion of genes for a transcription factor and a DNA binding dom ain . In a d d itio n to tum ors w ith t(2;13), the chim eric pro tein was also identified in an alveolar rhabdomyosar­coma cell line lacking this translocation, as w ell as in an und ifferen tia ted sar­coma . 69 Studies of other tum or cell lines, in c lu d in g em b ry o n a l rh ab d o m y o sar­coma, failed to demonstrate the presence of the PAX3/FKHR chim eric transcript. 69

Therefore, in small round cell tumors, th e p resen ce o f these ch im eric tran ­scripts as determ ined by PCR, may be of diagnostic utility, especially w hen cyto­genetics fail to demonstrate the charac­teris tic translocations. T hese may, in th e fu tu re , be im p o rta n t d iag n o stic criteria in defin ing tum or subtypes to guide therapy.

Summary

Im m u n o h is to lo g ic and cy togene tic studies are proving useful in the diagno­sis of many small round cell tumors. An aberran t cy togenetic profile of tum or ce lls and DNA p lo idy also p rov ides im portant prognostic information. In the future, m olecular studies, especially the pattern of tumor oncogene expression or the presence of chim eric transcripts, may also prove useful in diagnosis. For now, how ever, m olecu lar stud ies are com ­monly used only as a prognostic indicator for neuroblastom a. M olecular stud ies continue to elucidate histogenesis and re la tio n s h ip s am ong m any o f th e se tumors. This is most clearly seen to date in the triad of ES, PNET, and AT when contrasted with neuroblastoma. M olecu­lar techniques are also providing clues to help answ er such questions as why some tumors spontaneously regress.

Cytogenetics, im m unohistology, and m olecular studies are increasingly u ti­lized but by no means replace standard

clinical and laboratory diagnostic m odal­ities (including ligh t m icroscopy and electron microscopy) in the evaluation of the small round cell neoplasm s of childhood. Cytogenetics, imm unohistol­ogy, and m o lecu la r s tu d ie s p ro v id e inform ation that con tribu tes to tum or d iagnosis, som etim es to tum or p rog­nosis, and clearly adds to our u n d er­s tand ing o f m echan ism s invo lved in childhood neoplasia.

References

1. Triche TJ, Askin FB. Neuroblastoma and the differential diagnosis of small, round-, blue-cell tumors. Hum Pathol 1983;14:569-94.

2. Cavazzana AO, Magnani JP, Ross RA, M iser J, Triche TJ. Ewing’s sarcoma is an undifferenti­ated neuroectoderm al tumor. In: Evans AE, D ’Angio GJ, Knudson AG, Seeger RC, eds. Advances in Neuroblastoma Research II. New York: Alan R. Liss, 1988:487-98.

3. Ambros IM, Ambros PF, Strehl S, Kovar H, G adner H, Salzer-Kuntschik M. MIC-2 is a spe­cific marker for Ew ing’s sarcoma and periph­eral primitive neuroectoderm al tumors. Cancer 1991;67:1886-93.

4. F letcher JA. Cytogenetic aberrations in malig­nant soft tissue tumors. Adv Pathol Lab Med 1991;4:235-46.

5. D ehner LP. Primitive neuroectoderm al tum or and Ew ing’s sarcoma. Am J Surg Path 1993; 17: 1-13.

6. Seemayer TA, Vekemour M, de Chadarevian JP. Histological and cytogenetic findings in a m alignant tum or of the chest wall and lung (Askin tumor). Virchows Arch [Pathol Anat] 1985;408:289-96.

7. Becker LE, H inton D. Primitive neuroectoder­mal tumors of the central nervous system. Hum Pathol 1983;14:538-49.

8. D ehner LP. Pediatric Surgical Pathology. Bal­timore; Williams & Wilkins, 1987.

9. Beckwith JB. W ilm’s tum or and other renal tumors of childhood: a selective review from the National Wilm’s Tumor Study Pathology Center. Hum Pathol 1983;14:481-92.

10. Weeks DA, Beckwith JB, Mierav GW, Luckey DW. Rhabdoid tumor of kidney: a report of 111 cases from the Wilm’s Tumor Study Pathology Center. Am J Surg Pathol 1989;13:439-58.

11. Foschini MP, Van Eyken P, Brock PR, Casteels- Van D aele M, De Vos R, Dal Cin P, Van den Berghe H, D esm et VJ. M alignant rhabdoid tum or of the liver, a case report. Histopathol 1992;20:157-65.

12. Cattani MG, Viale G, Santini D, M artineli GN. M alignant rhabdoid tum or o f the uterus: an im m u n o h isto ch em ica l and u ltra s tru c tu ra l

76 W INTERS, GEIL, AND O ’CONNOR

study. Virchows Arch [Pathol Anat] 1992;420: 459-62.

13. Sola Perez J, Perez-Guillermo M, Bas Bermal A, M anzanera L opez T, Caro Lopez F. M alig­nant rhabdoid tum or of soft tissues: a cyto- pathological and immunohistochemical study. Diagnost Cytopathol 1992;8:369-73.

14. Sotelo-Avila C, Gonzalez-Crussi F, SadowinskiS, Gooch WM, Pena R. C lear cell sarcoma of the kidney: A clinicopathologic study of 21 patients w ith long-term follow -up evaluation . Hum Pathol 1985;16:1219-30.

15. Sonobe H, Mizobuchi H, M anabe Y, Furihata M, Iwata J, H ikita T, Oka T, Ohtsuki Y, Goto T. M orphological characteriza tion o f a new ly e stab lish ed hum an osteosarcom a cell line, HS-Os-1, revealing its d is tinc t osteoblastic nature. Virchows Arch [Cell Pathol] 1991;60: 181-7.

16. Bertoni F, Present D, Bacchini P, Pignatti G, Picci P, Campanacci M. The Istituto Rizzoli experience w ith small cell osteosarcoma. Can­cer ) 989;64:2591—9.

17. Ayala AG, Ro JY, Raymond AK, Jaffe N, Chawla SI, Carrasco H, Link M, Jim enez J, E deiken J, Wallace S, Murray JA, Benjamin R. Small cell osteosarcoma: a clinicopathologic study of 27 cases. Cancer 1989;64:2161-73.

18. Enzinger FM, Weiss SW. Soft Tissue Tumors. St. Louis: The C.V. Mosby Company, 1988: 936-45.

19. Knowles DM. N eoplastic Hem atopathology. Baltimore; Williams and Wilkins, 1992.

20. Kodet R. Rhabdomyosarcoma in childhood; an immunohistological analysis w ith myoglobin, desm in, and vim entin. Path Res Pract 1989; 185:207-13.

21. Cruz-Sanchez FF, Rossi ML, H ughes JT, Esiri MM, C oakham HB. M edu llob lastom a; an im m unohistological study of 50 cases. Acta Neuropathol 1989;79:205-10.

22. Abenoza P, Manivel JC, Wick MR, Hagen K, D ehner LP. Hepatoblastoma: an im munohisto­chemical and ultrastructural study. Hum Pathol 1987;18:1025-35.

23. Cheung NV, Saarinen UM, N eely JE, Land- m eier B, Donovan D, Coccia PF. Monoclonal antibodies to a glycolipid antigen on human neurob lastom a cells. C ancer Res 1985;45: 2642-9.

24. Kovar H, Dworzak M, Strehl S, Schnell E, Ambros IM, Ambros PF, G adner H. Overex­pression of the pseudoautosomal gene MIC2 in Ewing’s sarcoma and peripheral primitive neu­roectoderm al tumor. O ncogene 1990;3:1067— 70.

25. Shiniada H, Chatten J, Newton WA, Sachs N, Hanioudi AB, Chiba T, Marsden HB, Misugi K. H istopathologic prognostic factors in neuro- blastic tumors: definition of subtypes of gan- glioneuroblastoma and an age-linked classifica­tion of neuroblastomas. Natl Cancer Inst 1984; 73:40.5-13.

26. O p p ed a l BR, S to rm -M ath isen I, L ie SO, Brandtzaeg P. Prognostic factors in neuroblas­

toma clinical, histopathologic, and im m unohis­tochemical features and DNA ploidy in relation to prognosis. Cancer 1988;62:772-80.

27. Pilkinton GR, Pallesen G. Phenotypic charac­te r iz a tio n of non -h aem o p o ie tic sm all ce ll tumors of childhood w ith monoclonal antibod­ies to leucocytes, epithelia l cells, and cyto- skeletal proteins. H istopathology 1989;14:347-57.

28. Parham DM, W ebber B, H olt H, Williams WK, M aurer H. Im m unohistochem ical study of childhood rhabdom yosarcom as and re la ted neoplasms. Results of an intergroup rhabdo­myosarcoma study project. C ancer 1991 ;67: 3072-80.

29. Pinto A, Paslawski D, Sarnat HB, Parham DM. Im munohistochemical evaluation of dystrophin expression in small round cell tumors of child­hood. Mod Pathol 1993;6:679-84.

30. F e llin g e r EJ, G arin-C hesa P, G lasser DB, Huvos AG, Rettig WJ. Comparison of cell sur­face antigen HBA-71 (p 30/32 MIC-2), neuron- specific enolase, and vim entin in the im muno­histochemical analysis of Ew ing’s sarcoma of bone. Am J Surg Path 1992;16:746-55.

31. Oda H, Shiga J, M achinami R. C lear cell sar­coma of kidney, two cases in adults. Cancer 1993;71:2286-91.

32. D aim arv Y, H ashim oto H , T su n ey o sh i M, Enjoji M. Epithelial profile of epithelioid sar­coma: an im m unohistochem ical analysis of eight cases. Cancer 1987;59:134-41.

33. Arber DA, Kandalaft PL, Mehta P, Battifora H. V im entin-negative ep ithe lio id sarcoma: the value of an im munohistochemical panel that includes CD 34. Am J Surg Pathol 1993; 17: 302-7.

34. Mukai M, Torikata C, Iri H, Hanaoka H, Kawai T, Y akum aru K, S h im o d a T , M ika to l A, Kageyama K. Cellular differentiation of epithe­lio id sa rcom a: an e le c tro n -m ic ro sc o p ic , en zy m e-h is to ch em ica l, and im m u n o h is to ­chemical study. Am J Pathol 1985;119:44-56.

35. Fujii Y, Hongo T, Nakagawa Y, N asuda K, Mizuno O, Igarashi Y, Naito Y, M aeda M. Cell culture of small round cell tum or originating in the thoracopulm onary region. E v idence for derivation from a prim itive p luripo ten t cell. Cancer 1989;64:43-51.

36. Noguera R, Navarro S, Triche TJ. Translocation (11 ;22) in sm all cell osteosarcom a. C ancer G enet Cytogenet 1990;45:121-4.

37. Whang-Peng J, F reter CE, Knutsen T, Nanfro JJ, Gazdar A. Translocation t( ll;2 2 ) in esthesio- neuroblastoma. Cancer G enet Cytogenet 1987; 29:155-7.

38. Look AT, Hays FA, Nitschke R, McWilliams NB, Green AA. Cellular DNA content as a p re­dictor of response to chem otherapy in infants w ith unresectable neuroblastom a. N E ngl J Med 1984;311:231-5.

39. Fletcher JA, Kozakewich HP, Hoffer FA, Lage JM, W eidner N, T epper R, Pinkus GS, Morton

STUD IES O F SMALL ROUND C ELL TUMORS OF C H ILD H O O D 77

CC, Corson JM. Diagnostic relevance of clonal cytogenetic aberrations in m alignant soft-tissue tumors. N Engl J M ed 1991;324:436-42.

40. Lee W, Han K, Harris CP, Shim S, Kim S, Meis- ner LF. Use o f FISH to detect chromosomal translocations and deletions. Analysis of chro­m osome rearrangem ent in synovial sarcoma cells from paraffin-em bedded specimens. Am J Pathol 1993;153:15-9.

41. McGavran L, W aldstein G, Beckwith B, Collins J, Gilfillan S. Some consistent cytogenetic find­ings in Wilm’s tum or cells. Am J Hum G enet 1985;37:A32.

42. Yokoyama T, Tsukahara T, Nakagawa C, Kiku- chi T, M inoda K, Shim atake H. The N-myc gene product in primary retinoblastomas. Can­cer 1989;63:2134-8.

43. F letcher JA, Kozakewich HP, Pavelka K, G rief HE, Shamberger RC, Korf B, Morton CC. Con­sistent cytogenetic aberrations in hepatoblas­toma; a common pathway of genetic alterations in embryonal liver and skeletal muscle malig­nancies? Genes Chromosomes Cancer 1991;3: 37-43.

44. Cogen PH, Daneshvar L, M etzger AK, Edwards MS. D eletion m apping of the medulloblastoma locus on chromosome 17p. Genomics 1990;8: 279-85.

45. Chaganti RS, Klein EA. The cytogenetic basis for molecular analysis of leukem ia, lymphoma and solid tumor. In: Crossman J, ed. M olecular G enetics in C ancer D iagnosis. N ew York: Elsevier, 1990;73-104.

46. M olenaar WM, Dam -M eiring A, Kamps WA, C ornelisse CJ. DNA aneuploidy in rhabdo­myosarcomas as compared w ith other sarcomas of childhood and adolescence. Hum Pathol 1988;19:573-9.

47. Hata Y, Ishizu H, Ohmori K, Hamada H, Sasaki F, Uchino J, Inoue K, N aitoh H, Fujita M, Kobayashi T , Yokoyama S. Flow cytom etric analysis of the nuclear DNA content of hepato­blastoma. Cancer 1991;68:2566-70.

48. Shapiro DN, Parham DM, Douglass EC, W eb­ber BL, Newton WA, M aurer HM, Look AT. Relationship of tum or cell ploidy to histologic subtype and treatm ent outcome in children and adolescents w ith unresectable rhabdomyosar­coma. Proc Ann M eet Am Soc Clin Oncol 1990; 9:290.

49. Yasue M, Tomita T, Engelhard H, Ganzalez- Crussi F, M cLone DG, Bauer KD. Prognostic importance o f DNA ploidy in medulloblastoma of childhood. J Neurosurg 1989;70:385-91.

50. Look TA, Douglass EC, M eyer WH. Clinical importance of near-diploid tumor stem lines in patients w ith osteosarcoma o f an extremity. N Engl J M ed 1988;318:1567-72.

51. Schwab M, Ellison J, Busch M, Rosenav W, Varmus H E, Bishop JM. Enhanced expression of the hum an gene N-myc consequent to ampli­fication of DNA may contribute to malignant

progression of neuroblastoma. Proc Natl Acad Sci USA 1984;81:4940-4.

52. Garson JA, Clayton J, McIntyre P, Kemshead JT. N-myc oncogene amplification in rhabdo­myosarcoma at relapse. Lancet 1986; 1:1496.

53. Nisen PD, Zimmerman KA, Cotter SV, G ilbert F, Alt FW. Enhanced expression of the N-myc gene in Wilm’s tumors. Cancer Res 1986;46: 6217-22.

54. Leong PK, T hom er P, Yeger H, Ng K, Zhang Z, Squire J. D etection of MYCN gene amplifica­tion and deletions of chromosome lp in neuro­blastoma by in situ hybridization using routine histologic sections. Lab Inv 1993;69:43-50.

55. Nakagawara A, Ariama M, Azar C, Scavarda NJ, Brodeur GM. Inverse relationship betw een Trk expression and N-myc amplification in human neuroblastomas. Cancer Res 1992;52:1364-8.

56. Donovan MJ, H em pstead BL, Horvath C, Chao MV, Schofield D. Immunohistochem ical local­ization of Trk recep tor protein in ped iatric small round blue cell tumors. Am J Pathol 1993; 143:1560-7.

57. Nakagawara A, Arima-Nakagawara M, Scavarda NJ, Azar C, Cantor A, Brodeur GM. Association betw een high levels of expression of the Trk gene and favorable outcome in hum an neuro­blastoma. N Engl J M ed 1993;328:847-54.,

58. Bernards R, D esain SK, W einberg RA. N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastom a. Cell 1986;47:667-74.

59. Sugio K, Nakagawara A, Sasazuki T. Association of expression betw een N-myc gene and major histocompatibility complex class I gene in sur­gically resected neuroblastoma. Cancer 1991; 67:1384-8.

60. Akersen R, Bernards R. N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma. Mol Cell Biol 1990;10:2012-6.

61. Nakagawara A, Ikeda K, Higashi K, Sasazuki T. Inverse correlation betw een N-myc amplifica­tion and catecholamine metabolism in children with advanced neuroblastoma. Surgery 1990; 107:43-49.

62. Christiansen H, Christiansen NM, W agner F, A ltm annsberger M, L am pert F. N euroblas­toma: inverse relationship betw een expression of N-myc and NGF-r. Oncogene 1990;5:437-40.

63. Nakagawara A, Kadomatsu K, Sato S, Kohno K, Takano H, Akazawa K, Nose Y, Kuwano M. Inverse correlation betw een expression of m ul­tidrug resistance gene and N-myc oncogene in human neuroblastomas. Cancer Res 1990;50: 3043-7.

64. S tephenson C F , B ridge JA, Sandberg AA. Ewing’s sarcoma and neuroectoderm al tumors. Hum Pathol 1992;23:1270-7.

65. Bechet JM, Bornkamm G, Freese UK, Lenoir GM. The c-sis oncogene is not activated in Ewing’s sarcoma. N Engl J Med. 1984;310:393.

66. VanKessel AG, Turc-Carel C, deKlein A, Gros- veld G, Lenior G, Bootsma D. Translocation of

78 WINTERS, GEIL, AND O ’CONNOR

oncogene c-sis form chromosome 22 to chromo­some II in a Ewing’s sarcoma derived cell line. Mol Cell Biol 1985;5:427-9.

67. Lipinski M, H irsch MR, Deagostini-Bazin H, Yamada O, Tursz T, Goridis C. Characteriza­tion of neural cell adhesion molecules (NCAM) expressed by Ew ing and neuroblastom a cell lines. In t J Cancer 1987;40:81-6.

68. D elattre O, Zucm an J, M elot T, Garau XS, Zucker JM, Lenoir GM, Ambros PF, Sheer D,

Ture-Carel C, Triche TJ, Aurias A, Thomas G. The Ew ing family of tum ors-a subgroup of sm all-round-cell tumors defined by specific chim eric transcripts. N Engl J Med. 1994;331: 294-327.

69. Galili N, Davis RJ, Fredericks WJ, M ukhopad- hyay S, Rauscher FJ, Em anuel BS, Rovera G, Barr FG. Fusion of a fork head domain gene to PAX3 in the solid tum or alveolar rhabdomyo­sarcoma. Nat Gen 1993;5:230-4.