Small cell tumours in childhood: A review

JOURNAL OF PATHOLOGY, VOL. 145: 1-25 ( 1 985)

SMALL CELL TUMOURS IN CHILDHOOD: A REVIEW

S. VARIEND

Department of Histoputhology , Children’s Hospital, Shefield, U.K.

Received 20 July 1984 Accepted 24 July 1984

SUMMARY The past decade has seen significant advances in the treatment of childhood malignancies accompanied by appreci-

able improvement in survival rates. Treatment programmes have been largely formulated to meet the specific characteristics of individual tumours, as well as being based on the extent ofdisease presented at diagnosis. In selecting the most appropriate treatment protocol, accurate histological categorization of resected or biopsied tumour is thus of paramount importance. In the pacdiatric age range in which so many tumours lack differentiation as to present as, or mimic, other small cell tumours, routine methods are often insufficient to resolve problematic histology. A wide range of special techniques is now at hand to assist the pathologist with this problem and this review is an attempt partly to rationalize the application of available methodology. Of considerable importance also is a knowledge of the behavioural characteristics of this group of tumours, their prototypic histology, as well as the range of morphological variability. KEY WORDS- Childhood tumours, neuroblastoma, Ewing’s sarcoma, rhabdomyosarcoma.

INTRODUCTION

The recent substantial improvement in survival among children with malignant disease may be attributed largely to the introduction of multi- modality treatment and aggressive clinical staging of Histological diagnosis and extent of disease determine the choice of therapy protocol which, in turn, has increased the requirement for accurate pathological reporting. Consequently, imprecise diagnoses such as ‘small cell tumour’, otherwise unspecified, are unhelpful and should be strongly discouraged.

The new challenge for the histopathologist who is involved with childhood neoplasms is related particularly to neuroblastoma and rhabdomyosarcoma ; they commonly are poorly differentiated and may be easily confused histologically with other small cell neoplasms, notably Ewing’s sarcoma and non-Hodgkin’s lymphoma. To facilitate their distinction, there now exists a large battery of special diagnostic procedures that should be applied to their full advantage.

So that therapy may be instituted with the minimum of delay, speedy histological reporting is commendable, but the current practice of submit- ting smaller samples of tissue for histological assess-

c’ 1985 by John Wilcy & Sons, Ltd. 0022-341 7/85/0 1000 l-25$02.50

ment, especially since the advent of needle aspiration as a biopsy t e~hn ique ,~ has not particularly eased the situation. Over the past few years, however, the histopathologist has considerably widened his diagnostic armamentarium beyond that which rests on the assessment of simple two- dimensional morphology. Apart from the appreciable progress that has already been achieved with non-Hodgkin’s lymphoma, mainly through the harnessing of the well-known biological function of the lymphoid system, the list of tumour markers relating to other small cell tumours has also expanded rapidly. Such sophisticated methods should not, however, substitute for, but should serve rather to reinforce, conventional light microscopy. A sound basic understanding of the biological behaviour and nature of these neoplasm is, of course, also important as this undoubtedly contributes significantly towards their accurate evaluation.

NEUROBLASTOMA

Neuroblastoma is the third commonest solid malignant tumour of infancy and childhood, being superseded only by lymphoid neoplasms and

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cerebral tumours4~ Ninety per cent of tumours occur in patients less than 10 years of age,6although birth to 5 years represents the usual age range. There is a slight male preponderance. The tumour arises from neuroblasts, the undifferentiated pre- cursor cells of the sympathetic nervous system. The adrenal is the seat of primary growth in about a quarter of cases ;6 elsewhere, the neoplasm occurs mainly along the sympathetic chain. Consistent with this derivation from the sympathetic nervous system, neuroblastoma and its variants occur typically in a variety of paraspinal locations including the retroperitoneum, posterior mediastinum7 and cervical area,8 in order of decreasing frequency. Paralysis from tumours insinuating through inter- vertebral foramina causing spinal cord compression may O C C U ~ . ~ It is possible, however, that such dumb- bell neoplasms originate directly from the posterior root ganglia which are also of neural crest origin.’O Primary tumours occur exceptionally in the kid- ney’l and arise apparently from neural crest cells that have undergone aberrant migration.12 A staging system currently widely employed is that of Evans et d . 1 3 (Table I).

Of the common childhood tumours, neuroblastoma shows the most unpredictable clinical behaviour with a tendency to spontaneous ‘matura- tion’ or regression. l4 Patients with adrenal neuroblastoma generally have a poor outcome, related possibly to the high incidence of poorly differentiated growth at that site.1°

There is some evidence that patients with differentiating tumours fare better than those with undifferentiated neuroblastoma10 although general agreement concerning this association is still lacking.15 The greater proportion of favourably staged cases at a younger age is thought to account for the more favourable outcome among those under one year.16 The generally better prognosis among girls may be related to a greater capacity in that gender for the tumour to mature.6

Whilst bone marrow, liver and lymph nodal metastases are common, isolated pulmonary secondaries do occur but they are usually part of more widespread dissemination that constitutes a grave prognostic sign.I7 The outlook for ‘metastatic’ growth in the first year is not necessarily gloomy as many of these cases frequently undergo spontaneous resolution of their disease, an outcome which is particularly associated with Stage IV-S cases.12 Compared with conventional Stage VI, patients with Stage IV-S disease have an excellent prognosis even without treatment although it may

Table I-Neuroblastoma staging

I Tumour confined to structure of origin I1 Tumour extends beyond structure of origin but in

continuity and not crossing the midline. (Regional lymph nodes on the same side may be involved)

111 Tumour extends in continuity beyond midline. (Lymph nodes may be involved on both sides)

IV Distant disease I or I1 and distant disease, e.g. liver, skin or bone marrow (but without x-ray evidence of bone involvement)

IV-S

take some time for hepatic enlargement and skin nodules to regress. The occurrence of Stage IV-S disease in the neonate, however, seems to be an exception to this excellent prospect for survival.18

Clusters of neuroblasts may persist in the adrenal medulla after birth and be found incidentally at autopsy, usually in infants less than three months of age.19,20 These lesions are referred to as neuroblastoma-in-situ, the incidence of which has been calculated to be about 40 times greater than clini- cally overt neuroblastoma, suggesting that many such lesions involute with time. According to Knudson’s two-hit theory of oncogenesis,2’ neuroblastoma-in-situ may be considered an expression of the first hit with a second hit manifest- ing as overt neuroblastoma. Stage IV-S disease, with its high incidence of spontaneous regression, has been considered in a similar contextZ2 and the possibility that it represents a multifocal lesion related to abnormally placed neural crest cells has been considered. lo

Histologically, the tumour seems committed to a programme of continuous differentiation with progressive neurogenesis giving rise to a histological spectrum that includes neuroblastoma, ganglioneuroblastoma and ganglioneuroma. Undifferenti- ated neuroblastoma presents a structureless array of small, round to ovoid cells (Fig. I ) , frequently divided into lobules by delicate, vascular connective tissue septiae. Nuclei are hyperchromatic but mitoses are not particularly common. Pseudo- rosettes and patches of pink fibrillary material are useful histological markers of early differentiation (Figs 2a, b) while early neuronal development is characterized by increasing nuclear vesiculation, prominent nucleoli and more voluminous cytoplasm (Fig. 2b). Necrosis and focal calcification are common.

The boundary between neuroblastoma and ganglioneuroblastoma is ill-defined’ with no general agreement on the degree of neural differentiation

SMALL CELL TUMOURS IN CHILDHOOD 3

Fig. I - Sheets of small cells with scant cytoplasm and hyperchromatic nuclei with no tendency towards organoid differentiation are the usual features of poorly differentiated neuroblastoma. H & E, x 314

that separates these two groups. A composite pattern, characterized by one or more discrete nodules of pure neuroblastoma occurring in a ganglioneuroblastoma, and a diffuse pattern which shows a mixture of primitive and differentiating neuroblasts, further categorize ganglioneuroblast~rna.~~~~ The importance of this distinction lies in the better outlook for patients with the diffuse pattern.

Ganglioneuromas are benign, often encap- sulated, generally circumscribed tumours that may attain an enormous size.7 They have an anatomical distribution similar to that of neuroblastomas. Foci of cystic degeneration, haemorrhage and calcification are seen occasionally. Microscopically, relatively mature ganglion cells occur singly or in groups set in an abundant neurocollagenous matrix (Fig. 3). Satellite cells may or may not be present. Ganglion cells may retain a degree of multi- nuclearity but mitoses are always absent. Multiple sections should be examined in order to exclude residual neuroblasts which, however, should not be mistaken for lymphocytic foci that are fairly common.

Biochemical aspects Many of these tumours are biologically active

and capable of elaborating a variety of

catecholamine precursors and their metabolite^.^^ Vanilylmandelic acid (VMA), homovanillic acid (HVA), or both, are secreted in 80 per cent of patients with neuroblastoma.16 Noradrenaline and adrenalin, as such, are rarely secreted and urinary VMA and HVA are usually measured as biochemical markers to aid diagnosis and monitor therapy. Hypertension may occur, probably related to the secretion of these compoundszs although, with retroperitoneal tumours, renal pedicle compression as a mechanism should also be c o n ~ i d e r e d . ’ ~ ~ ~ ~ The ratio of VMAIHVA is also regarded a useful prognostic index, higher values being associated with a more favourable outcome.z6 Some tumours, particularly those with advanced differentiation, produce and secrete VIP with intractable diarrhoea as the main clinical presentation.z7

Ancillary diagnostic methods

Touch-imprints of tumour stained with haematoxylin and eosin show characteristic small groups of tumour cells with tapering cytoplasmic extensions directed radially or linking individual cells, and may be present even when differentiation is lacking in routine preparations (Fig. 4). Glyoxylic acid-induced fluorescence of tumour cells has been reported to occur in over 80 per cent of cases.z8

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Fig. 2-(a) Neuroblastoma with well developed rosettes containing central neurofbrillary cores. H & E, x 502. (b) On the right side of the field there is poorly nucleated neurofibrillary material; the left shows a mixturc of neuroblasts and larger cells with features of early neurogenesis. H & E, x 314

SMALL CELL TUMOURS IN CHlLDHOOD 5

Fig. 3-GanghOneUrOmd showing clusters of ganglion cells set in a stroma of neurocollagenous tissue. H & E, x 125

Other diagnostic aids include the utilization of monoclonal antibodiesz9 and immunochemical markers such as neurone-specific enolase30 which is not specific, however, as a similar reaction may occur in other tumours such as melanoma.

The unique property of tumour cells to form neurite processes and cell clumping in tissue cul- ture has been exploited as a diagnostic

method.28 Neuroblastoma has recognized status among the neuroendocrine tumours3 and, apart from its ability to produce catecholamine, is also known to elaborate acetylch~linesterase.~~ The presence of this enzyme has been demonstrated by histochemistry in human neurob la~ toma~~ (Fig. 5 ) . Such reactivity for acetylcholinesterase does not appear to occur in Ewing’s sarcoma or lympho-

Fig. 4 Touch-imprint of poorly differentiated neuroblastoma Fig. 5-Poorly differentiated neuroblastoma with strong showing frequent cytoplasmic extensions, sometimes bridging cytoplasmic positivity for acetykholinesterase. Acetyl- individual tumour cells. H & E, x 1056 cholinesterdse, x 264

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blastic lymphoma although, with rhabdomyosarcoma, focal positivity may be demonstrated. Glycogen, as demonstrated by diastase-sensitive PAS-positivity, is present in a large proportion of n e u r o b l a s t ~ m a s ~ " ~ ~ but the en bloc positivity encountered in rhabdomyosarcoma and Ewing's sarcoma has not been reported.26

Ultrastructure

Catecholamine granules measuring 50-200 nm in diameter are usually regarded as fundamental to ultrastructural d i a g n ~ s i s . ' ~ ~ ~ ~ - ~ ~ The granules, which should be distinguished from lysosomes, are present in the cell body but are generally more frequent in the cytoplasmic extensions3' (Fig. 6). A correlation between the level of urinary catecholamine products and the frequency of occurrence of catecholamine granules has been reported.3s Increasing tumour differentiation also appears to be associated with greater numbers of catecholamine granules.38 A compact meshwork of interdigitating neurites containing longitudinally orientated neurofilaments and neurotubules (approximately 20 nm in diameter (Fig. 7)) is common and useful in d i a g n o s i ~ , ~ ~ ~ ~ ~ and their number and length increase proportionately with degree of

tumour differentiation, being particularly pronounced in gang l ione~roma .~~ Clear vesicles, about half the size of catecholamine granules, probably representing acetylcholine activity, may be p r e ~ e n t . ~ ~ , ~ ~ Cell-cell attachments and glycogen may be seen.ls Mitochondria, granular and smooth endoplasmic reticulum, free ribosomes and Golgi complexes are other features whose numbers appear to increase in accordance with the degree of neurogenic differentiation.

Related neoplasms

Neuroblastoma containing neuromelanin has been reported.40 A special variant of neuroblastoma derived from the olfactory apparatus, so- called olfactory neuroblastoma (esthesioneuroblastoma), resembles conventional neuroblastoma both histologically and ultrastructurally.41~4z Although the majority of these tumours has been reported in adults, a modest number occur in children.

Neuroblastoma also occurs as a component of progonomas (melanotic neuroectodermal tumour of infancy)43 where it forms part of a biphasic pattern in conjunction with melanin-containing cuboidal cells. Generally, patients are under 1 year

Fig. 6-Two transversely sectioned neurites reveal dense, cored, neurosecretory granules (arrow). Uranyl acetate & lead citrate, x 68 400


Fig. 7-Neuroblastoma showing intertwining, closely apposed dendrite processes. Uranyl acetate & lead citrate, x 6840

of age and, although the great majority of cases affect the maxilla, they have been described occasionally in the mediastinum, epididymis and long bones. Rarely, the neuroblastomatous component may predominate and mask the underlying pattern.

N e ~ r o e p i t h e l i o m a ~ ~ ~ is a rare malignant tumour with microscopic and ultrastructural features resembling neuroblastoma. These include the presence of pseudo-rosettes (Fig. S), and an ability in some to elaborate catecholamine metabolite^.^^ It is probable that such tumours represent neuroblastoma arising in peripheral nerves. Neuro- epitheliomas are more commonly reported in adults but their relation to the small cell tumours of the chest wall in children and adolescents, as described by Barson et d 4 I and Askin et U I . ~ ~ remains ill- defined. So-called Askin tumours have been shown recently to immunoreact for neurone-specific e n ~ l a s e , ~ ~ as have similar tumours affecting

RHABDOMYOSARCOMA

one year of age observed in other small cell tumours is a feature not shared by rhabdomyosarcoma. 5 1 9 5 2

The extent of disease and degree of resectability determine the stage of diseases3 (Table 11).

Microscopic diagnosis depends on pattern recog- nition and evidence of skeletal muscle differentiation, notably the presence of rhabdomyoblasts and the demonstration of cytoplasmic cross-striations.

Rhabdomyosarcoma is the commonest soft tis-

group is generally associated with better Survival, the particularly excellent outcome of children under

sue Sarcoma in the younger age Fig. 8-Neuroepithelioma from the chest wall of a 12 year-old boy showing pseudo-rosettes reminiscent of neuroblastoma. H & E, x 256

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Table I 1 - Rhabdomyosarcoma staninn

I Disease localized- completely resected I 1 Regional disease, resected I11 Localized disease not completely resected IV Metastatic disease presenting at diagnosis

Cross-striations, now generally regarded as not essential for diagnosis, are recognized to occur in less than 50 per cent of turn our^.^^^^^ Rhab- domyoblasts exhibit abundant pink-staining, slightly granular cytoplasm that may assume a variety of shapes; strap, tadpole, oval, spindle and racquet; the cytoplasm stains deep red with Mas- son’s trichrome and deep blue with PTAH.55 Cross- striations, when present, are found within the cytoplasm of the rhabdomyoblast. The histological categories of rhabdomyosarcoma include the embryonal, alveolar and pleomorphic patterns.56

Embryonal tumours predominate in the head, neck and genitourinary tract, including the epididymis; other well recognized, but less common sites are the thorax, biliary tree, retroperitoneum and perineum. Males are affected more commonly and the average age of presentation is 3 4 years. Congenital tumours have been described. 5 7 , 5 8

Embryonal rhabdomyosarcoma tends to arise

where striated muscle does not normally occur and origin from uncommitted multipotent primitive mesenchyme seems probable.59

Neoplasms of the head and neck are generally associated with poor survival compared with those arising in the genito-urinary tract. This less favourable outlook for the head tumours, in particular those arising in the nasopharynx, paranasal sinuses and middle ear, has been ascribed to their parameningeal location and, consequently, early infiltration of the central nervous system.60

The embryonal sub-group may be further cate- gorized into those showing predominantly a myxoid pattern, spindle-cell pattern or a round-cell pattern.61 The myxoid pattern is comprised of stellate and spindle cells arranged in a loose syncytium and set in a mucoid matrix (Fig. 9); nuclei are hyperchromatic and mitoses are generally frequent.

Although essentially an embryonal tumour, the botryoid rhabdomyosarcoma, which grows as a grape-like cluster beneath the mucous membrane of hollow viscera (Fig. lo), has been regarded as a special group.62 This form of the tumour predominates in the genito-urinary tract where it involves the trigone of the bladder, the anterior vaginal wall in females and the prostate in males, but may also arise in the middle ear, maxillary sinus,

Fig. 9 - Myxoid type of embryonal rhabdomyosarcoma showing small, dark, stellate cells set in a loose mucoid background H & E , x314


Fig. 10- Botryoid-embryonal rhabdornyosarcoma from the middle ear of a 4 year-old boy showing typical, grape-like, polypoid appearance. x 2.8

nasopharynx and soft palate.56 It is the usual type seen in association with the biliary tract.50 Histolo- gically, it is characterized by a dense zone of sub- epithelial neoplastic cells, the so-called cambium layer, which frequently reveals rhabdomyoblasts while the deeper portions are more vascular, myx-

oid and relatively acellular (Fig. 1 I).,, The round cell pattern (Fig. 122) may be easily

confused with a variety of other small cell tumours, although early rhabdomyoblasts are revealed frequently after diligent searching. The spindle-cell pattern of rhabdomyosarcoma (Fig. 13) resembles fibrosarcoma, fibrous histiocytoma or leiomyo- sarcoma while myxoid liposarcoma and chondrosarcoma should be considered when the myxoid pattern prevails.63 On the other hand, many of these tumours are unusual in childhood and need not be considered ordinarily in the differential diagnosis of rhabdomyosarcoma in this age group.

Benign vaginal polyps with features similar to the botryoid pattern of rhabdomyosarcoma have been reported and, although the majority occur in older subjects, infants are not exempt.64 Fetal rhab- d o m y ~ m a ~ ~ v ~ ~ a benign tumour usually located in the subcutaneous soft tissue, has a similar age distribution to that of embryonal rhabdomyosarcoma and presents a potential source of diagnostic con- fusion. The head and neck are usually affected with predilection for the posterior auricular area. A marked male predominance exists. These lesions are generally circumscribed and histologically feature a mixture of rhabdomyoblasts and primitive mesenchymal cells ; rhabdomyoblasts occur com-

Fig. 11 -Microscopic view of a botryoid tumour showing condensation of tumour cells beneath attenuated epithelium (cambium layer). The deeper part is less cellular and more myxoid in appearance. H & E, x 216

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Fig. 12-A round cell variant of embwonal rhabdomyosarcoma. Biopsy of orbital tumour from a 10 month-old female. H & E, x415

monly at the tumour periphery. Compared with rhabdomyosarcoma, the cell population is more uniform, hyperchromatic cells are lacking and mitoses are rare or absent.

Alveolar rhabdomyosarcoma is usually recognized by a pattern of irregular spaces or ‘alveoli’ separated by vascular fibrous septa of variable thickness.ss~58~66 The spaces are lined by a layer of tumour cells that impart an epithelial appearance and may show degrees of myoblastic differentiation. The spaces contain dehiscent or ‘floating’ cells that are frequently pyknotic and karyhorrectic (Figs 14a, b). Characteristic multinucleated tumour giant cells with a wreath of marginally placed nuclei are fairly (Fig. 15). Mitotic figures may be sparse. The alveolar pattern may be lacking, however, and coexist with extensive areas in which the cells are closely packed. The alveolar spaces develop probably as a degenerative feature and, consequently, are found more often in the centre of the tumour where vascularization is likely to be most precarious. Multiple sections may be necessary therefore to disclose this characteristic.

Compared with the embryonal sub-group, alveolar rhabdomyosarcoma is generally associated with a less favourable The incidence of alveolar rhabdomyosarcoma increases with age and

there is a predilection for the soft tissues of the e x t r e m i t i e ~ . ~ ~ This pattern of the tumour is not limited to skeletal muscle, however, and there is some overlap with the usual anatomical distribution of embryonal tumours with periorbital, perianal and perineal soft tissues being other recognized primary sites. 54 Alveolar rhabdomyosarcoma tends to metastasize to lymph nodes, lungs, pleura, pancreas and boness and diffuse lymph nodal and bone marrow metastases with a leucoerythroblastic blood picture, which may stimulate a haemopoietic malignancy, is a recognized clinical presentation.68 In such cases, the primary tumour may not be discovered even after exhaustive searching. That embryonal and alveolar patterns may co-exist in the same tumour is well recognized ;51, 57 ,69 this pattern has been referred to as mixed rhabdomyosarcoma. s8

The embryonal and alveolar patterns resemble developing muscle during early gestation.69 The pleomorphic pattern, on the other hand, which predominates in adults does not seem to recapitulate any recognizable stage of muscle development and probably represents dedifferentiation in mature striated muscle.69 The pleomorphic pattern seems to be rare in childhood with a reported frequency of 0.7 per cent and 5 per cents7 However, many of the

SMALL CELL TUMOURS IN CHILDHOOD 1 1

Fig. 13-Spindle-cell variant of embryonal rhdbdomyosarcoma with focal rhabdomyoblastic differentiation. H & E, x 195

documented large series have not included its occur- r e n ~ e , ~ l , 5 8 and such disparity is possibly related to the application of different diagnostic criteria.

Soft tissue tumours lacking sufficient cellular differentiation to allow an unequivocal histological diagnosis of rhabdomyosarcoma have been vari- ably referred to as ‘sarcoma of undetermined histogenesis’,58 ‘undifferentiated mesenchymal sarcoma’55 and ‘mesenchymal sarcoma, probably rhabdomyosarcoma’. 5 7 In practice, the clinical response of these neoplasms to the usual therapeutic regime advocated for proven rhabdomyosarcoma has been similar.70 However, there now exists a wide range of special techniques that may aid substantially in resolving the histology of tumours presenting diagnostic difficulty by routine methods.

Histochemistry and immunochemistry

Positive staining for glycogen is common in rhabdomyosarcoma and its successful demonstration is enhanced in frozen sections (Fig. 16), or by prior fixation in absolute alcohol. PTAH staining is useful and serves to emphasize cytoplasmic cross- striations frequently already apparent on conventional staining.

Myosin may be demonstrated histochemically in tumour cells using the myofibrillar (calcium- activated) adenosine triphosphatase technique (Fig. 17).71972 An advantage of this method is that it can be carried out rapidly and the reagents used are generally inexpensive. Both early and advanced rhabdomyoblasts are accentuated with the method carried out following acid pre-incubation. At this pH, the small undifferentiated cell of alveolar rhabdomyosarcomas also reacts with a degree of positivity that easily distinguishes the cell type from other small celled tumours likely to be encountered in the paediatric age range.59

Myophosphorylase, a glycolytic enzyme present normally in striated and smooth m ~ c l e , ~ ~ may also be demonstrated histochemically in rhabdomyo- ~ a r c o m a , ~ ’ . ~ ~ but a positive reaction is probably limited by the degree of cyto-differentiation. Rhab- domyoblasts also reveal focal acetylcholinesterase activity (Fig. IS), a feature that is probably related to the early development of acetylcholine neuro- transmitter activity.

Immunochemical diagnosis of rhabdomyosarcoma has utilized antisera directed against a variety of muscle proteins including myosin,74 a ~ t i n , ~ ~ d e ~ m i n ’ ~ and my~globin.~’ 8o However, only myoglobin appears to be specific for skeletal

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(h)

Fig. 14--(a) Rhabdomyosarcoma showing a well defined ‘alveolar’ pattern. The spaces are separated by thick fibrous bands and lined by tumour cells; they contain degenerate, ‘free-floating’ cells. (b) The ‘alveolar’ pattern is less developed in this tumour. H & E. x 314


Fig. 15--Alveolar rhabdomyosarcoma containing several tumour giant cells with peripherally marginated nuclei. H & E, x 125

Fig. 16- Embryonal tumour metastic to lymph node showing strong, frequently clumped, PAS cytoplasmic positivity. PAS, x 314

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Fig. 17-Small and large rhabdomyoblasts are heavily accentuated by the ATP-ase reaction in an embryonal tumour. ATP-ase with acid pre-incubation, x 205

muscle as the remainder occur in other tissues, most notably smooth muscle. Thus, the demonstration only of immunoreactive myoglobin indicates that the tumour is truly rhabdomyoblastic in nature (Fig. 19); yet studies on its diagnostic effectiveness have, so far, fallen far short of 100 per ~ent , ’~-~O regardless of histological type, although a positive reaction is more frequent with alveolar rhab- do rnyosa r~oma .~~ Failure of tumour cells to react positively may be due to lack of differentiation of the tumour, or result from sub-optimal fixation of tissue.

Caution in interpretation is necessary when undifferentiated tumour occurs in or near normal striated muscle as deeply entrapped non-neoplastic myofibres are likely to immunoreact for myoglobin and lead to erroneous diagnosis. Cells of non- rhabdomyosarcomatous tumours metastic to skeletal muscle as well as local reactive histiocytes may react positively for myoglobin, apparently due to the ‘uptake’ of myoglobin released from dam- aged skeletal muscle.s1 p-Enolase, a dimeric glycolytic enzyme found normally in striated muscle, has also been shown recently to have a high degree of specificity as an immunomarker of rhabdomyosarcoma.82

Ultrastructure

The value of electron microscopy lies mainly in its potential to confirm the presence of rhabdomyoblastic differentiation.8s86 Rhab- domyoblasts are likely to be confused at the light microscopy level with the cells of malignant

Fig. 18 --Strong, patchy, acetylcholinesterase cytoplasmic reactivity in embryonal rhabdomyosarcoma. Acetylcholinesterase, x 264

melanoma or epithelioid sarcoma and, arguably, electron microscopy has greater applicability in an older age group where such tumours are much more common. The minimal ultrastructural criteria advocated for confirmation of rhabdomyoblasts are the presence of alternating thick (myosin) filaments and thin (actin) filaments arranged in b ~ n d l e s . ~ ~ . s ~ Intermittent formation of attentuated Z-band material, occurring as rectangular densities with their long axes perpendicular to the alignment of filaments is an additional helpful featureE7 (Fig. 20). Such appearances are an attempt at sarcomere organization, the fundamental contractile unit of normal skeletal muscle. The small primitive cell of rhabdomyosarcoma, on the other hands, shows

Fig. 19-Turnour cells of embryonal rhabdomyosarcoma show focal reaction for myoglobin. Imrnunoperoxidase, x 422


Fig. 20-Cytoplasm of tumour cell in an alveolar rhabdomyosarcoma showing bundles of alternating thick and thin filaments with occasional rectangular densities (arrow). Uranyl acetate and lead citrate. x 40 850

monoparticulate glycogen, pinocytotic vesicles, cell junctions and basement membrane which, essentially, are non-specific and, not unexpectedly, may accompany sarcomeric organization in the better differentiated tumour. Thus, when rhabdomyosarcoma is completely undifferentiated by light microscopy, the value of electron microscopy with respect to yield of confirmatory evidence, is arguable, a problem undoubtedly exacerbated by the inherent problem of sample limitation.

Compared with the embryonal tumours, tumour cell junctions in alveolar rhabdomyosarcoma are common while myofilament formation in such tumours is relatively less advanced.84 At certain sites, rhabdomyosarcomas may incorporate adja- cent normal muscle fibres and particular caution is necessary at the ultrastructural level to distinguish these from neoplastic cells. Such entrapped, non- neoplastic muscle fibres are, however, generally recognized by a degree of myofilamental organization comparable to that found in normal muscle.

NON-HODGKIN’S LYMPHOMA AND LEUKAEMIA

Non-Hodgkin’s lymphoma (NHL) and leukaemia account for a significant proportion of

malignant tumours among children under the age of fifteen In children, NHL differs from that occurring in adults insofar as they are predominantly extranodal, rarely nodular, often leukaemic, rapidly proliferative and they rarely secrete para protein^.^^,^^ Also, there is usually an association between the different histological types and anatomical site and the clinical response rate to therapy is e n c o ~ r a g i n g . ~ ~ The majority of childhood NHL fall into four major histological categories : lymphoblastic lymphoma, Burkitt’s lymphoma, undifferentiated lymphoma and large- cell (‘histiocytic’) type.88,89 In view of the predominance of extranodal disease, non- contiguous involvement and frequent progression to leukaemia, the Ann-Arbor staging system is diffi- cult to apply and other systems have been pro-

Spread to the central nervous system is common and represents a poor prognostic fea- t ~ r e . ~ ~ The great majority of childhood NHL is committed to T- or B-cell differentiation, contrast- ing with the usual phenotype of most childhood lymphoblastic l e ~ k a e m i a s . ~ ~

Lymphoblastic lymphoma accounts for approximately 3 5 4 0 per cent of childhood NHL, tends to predominate in the supra-diaphragmatic region and presents commonly as a mediastinal mass (Fig. 21)88,89 Males are affected more commonly

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than females and the peak age incidence is about 1 I years. Morphologically, lymphoblastic lymphoma and acute leukaemia (ALL) are indis- tinguishableg3 and, though certain differences exist in their clinical behaviour, i t is increasingly evident that they express stages of the same neoplastic process.94

Histologically, lymphoblastic lymphoma is characterized by a diffuse, monomorphous, small cell pattern with round to ovoid nuclei, and a rim of barely discernible cytoplasm (Fig. 22).95 Chromatin is finely dispersed and two to several nucleoli are present. Nuclear convolutions may be numerous, sparse or absentg6s9’ and the mitotic count is usually high. The presence of macrophages containing ingested cellular debris frequently imparts a ‘starry sky’ pattern, although this feature is not usually as pronounced as in Burkitt’s lymphoma or undifferentiated lymphoma. Diastase- digestible PAS-positive staining for cytoplasmic glycogen is sometimes present but the clumped positivity seen in Ewing’s sarcoma and rhabdomyosarcoma has, so far, not be described.26 Methyl green pyronine staining is minimal and Fig. 21-Chest radiograph showing a mediastinal mass in a 12

year-old boy with lymphoblastic lymphoma (courtesy of Dr R. K . Levick) chloroacetate esterase.

turnour cells are negative for

Over a wide age range the majority of lym-

Fig. 22-Lymphoblastic lymphoma showing monomorphic appearance of tumour cells and vigorous mitotic activity. Several macrophages are also present and impart a ‘starry sky’ effect. H & E, x 523


phoblastic lymphoma are of T-cell origin98 and are also positive for terminal deoxynucleotidyl transferase (TdT).97 A modest number belong to the non-B, non-T group or are of B-cell lineage.93 Punctate paranuclear positivity with the acid phosphatase reaction is a useful histochemical marker (Fig. 23).

Sporadic non-African Burkitt’s lymphoma (small, non-cleaved cell malignant lymphoma) is morphologically indistinguishable from endemic African Burkitt’s lymphoma99 and has a notable predilection for the abdominal viscera.96 The tumour is composed of cells that are of uniform size with a thin rim of basophilic cytoplasm and round- to-ovoid nuclei containing coarse chromatin.94 Nuclear convolutions are absent and 2-5 eosinophilic nucleoli are fairly conspicuous. Mitotic activity is pronounced and a ‘starry sky’ pattern, due to the presence of macrophages frequently containing ingested cell debris, is usually appreciable. Fine cytoplasmic vacuoles which stain positively for neutral fat can be discerned with high magnification in thin sections or imprint preparations. Cytoplasm is strongly pyroninophilic. PAS staining is con- sistently negative and there is no naphthol-ASD chloroacetate esterase activity. Burkitt’s lymphoma has surface immunoglobulin and other characteristics of a B-cell neoplasm. loo

Undifferentiated lymphoma, also a B-cell tumour, occurs predominantly in adultsIo’ but a limited number of cases have been reported in children.Io2 The anatomical distribution is similar to that of Burkitt’s lymphoma. The cells are larger and more pleomorphic, nucleoli are fewer but more distinct. Nuclear chromatin is coarse. The histochemical features, however, are similar to those of Burkitt’s lymphoma and cytoplasmic vacuoles may be also demonstrated. lo’ The value of distinguish- ing undifferentiated lymphoma from Burkitt’s lymphoma lies in the poorer prognosis of the former.’02

Large cell lymphoma (LCL), an immunologically heterogeneous group, is now recognized to arise mainly from transformed lymphocyte^.'^^ A number of reports have documented their occurrence in children.93- lo2> Io4 Compared with the preceding groups, the anatomical distribution of LCL is much more random. Presentation as a mediastinal mass is rare while central nervous system, hepatic and bone marrow involvement is unusual at the time of diagnosis.88 More than 50 per cent of such tumours are derived from B-cells and 5-10 per cent have T- cell markers ; a small proportion are truly histiocytic or have no detectable markers.Io3 Histologically,

Fig. 23-Dot-like paranuclear positivity with acid phosphatase reaction in lymphoblastic lymphoma. H & E, x 1056

true histiocytic tumours are recognized by cells with large irregular vesicular nuclei, prominent nucleoli and abundant, weakly eosinophilic cytoplasm. Their histiocytic derivation may be confirmed by positive staining for lysozyme, a-antichymotrypsin, non-specific esterase or acid phosphatase.Io5

Granulocytic sarcoma (chloroma) This uncommon tumour, composed of granulo-

cyte precursors,lo6, lo’ is associated with and frequently precedes the onset of leukaemia, usually of the acute myeloid type. Bone, orbit, soft tissue, lymph node and skin are common sites and there is a predilection for the tumour to occur in childhood. Well-differentiated tumours contain eosinophil precursors that are an important clue to diagnosis, and when poorly differentiated, the tumour is most commonly misdiagnosed as lymphoma. Naphthol-ASD chloroacetate esterase and antilysozyme immunoperoxidase staining are positive in the majority of cases and are an important aid to diagnosis.

EWING’S SARCOMA

In 1921 James Ewinglo8 described a primary malignant tumour of bone in which he emphasized the perivascular arrangement of the tumour cells and suggested a derivation from vascular endo- thelium. Any age or bone may be affected but the neoplasm predominates in the second decade of life and the pelvis and femur are the most common sites.

18 S. VARIEND

In one large study, 2 per cent of tumours occurred in babies and toddlers.'09 Pain and swelling are common symptoms and there is a modest male preponderance. Compared with distal lesions, tumours arising in the pelvis and proximal extremities tend to be associated with a lower survival

The histogenesis is still a matter of controversy and, apart From the suggested endothelial cell origin, Ewing's sarcoma has been also considered to be derived from the primitive mesenchymes4 or myelogenous tissue.112 The rare co-existence of Ewing's sarcoma and myeloid leukaemia has been used as argument against the latter thesis,lI3 while the occasional location of histologically similar tumour in soft tissue114 may be viewed as support- ing a derivation from mesenchyme.

Ewing's sarcoma has a strong propensity to penetrate cortical bone and develop an extra- osseous mass, often far removed from the epicentre of the tumour, which is commonly the metaphysi~."~ Resected tumour is white and soft, presumably due to lack of appreciable stroma, or may be yellow and friable with advanced degeneration. Metastases to lung, bone and regional lymph nodes are common but, with longer survival from

rate. 1 10,111

more effective therapy, cerebral metastasis is recognized increasingly.

The tumour is extremely friable and frequently shows necrosis and degeneration.111.113g 116 In the better preserved areas the cells are moderate in size with generally round to oval and vesicular nuclei. Nuclear membranes are distinct and nucleoli are inconspicuous. Mitoses are present in most tumours but are not usually abundant. Cytoplasm is finely granular, but may be clear or vesicular, reflecting presumably the presence of intracellular glycogen. Cell boundaries are typically ill-defined. A proportion of tumours show cells with small, dark, elongated nuclei and scalloped cytoplasmic borders. These cells occur singly or in groups (Fig. 24) and are commonly referred to as 'secondary cells'. Although their nature is still controver- sial, a degenerative origin is favoured by most.117-119 Aggregation of cells around necrotic foci is the explanation advanced for the rosettes encountered occasionally in Ewing's sarcoma;1'6compared with neuroblastoma, the rosettes of Ewing's sarcoma are more irregular in outline and lack a distinct neurofibrillary core. A range of histological patterns including diffuse, lobular, filigree and trabecular arrangements are recognized." Those tumours

Fig. 24- Ewing's sarcoma showing a dimorphic pattern. The principal cells have vesicular nuclei with well-defined nuclear membranes and poorly delimited cytoplasmic boundaries. The nuclei of the secondary cells are, by contrast, small, dark and angulated. The tumour is divided irregularly into lobules by delicate vascular connective tissue septa. H & E, x 201


showing a filigree pattern are reported to be associated with the least favourable outcome. A propensity for some tumours to invade the sarcoplasm of skeletal myofibres has been also viewed as a poor prognostic sign. lZo

Diatase labile PAS-positive staining for glycogen in tumour cell cytoplasm is common, the positivity occurring en bloc or finely dispersed (Fig. 25). The glycogen content of the ‘secondary cell’ is sparse by comparison, or may be absent. The demonstration of glycogen is influenced by the type of fixation, and optimal results are usually obtained with prior fixation in absolute alcohol or in frozen sections. Characteristically, staining for reticulin shows fibres related to connective tissue or vascular walls with little tendency to encompass individual tumour cells (Fig. 26).115,116 Cell membrane-bound alkaline phosphatase activity has been demonstrated.

Ewing’s sarcoma may mimic osteomyelitis clini- cally, radiologically and even on histology. IZ1, 122

Histologically, the tumour should be distinguished from the round cell variant of o ~ t e o s a r c o m a ~ ~ ~ and mesenchymal chondrosarcoma (poly- h i s t i ~ m a ) . ’ ~ ~ ~ ~ ~ ~ The latter neoplasm may display cytoplasmic glycogen but, on careful searching, foci of chondroblastic differentiation are frequently revealed. Mesenchymal chondrosarcoma spans a

wide age range with the highest incidence in the second and third decades ; occasionally, younger patients are affected. The primary tumour may involve bone or soft tissue. In the small cell variant of osteosarcoma, reticulin staining shows fibres encompassing individual tumour cells.

Soft tissue Ewing’s sarcoma

Several reports have documented the existence of a soft tissue or extra-skeletal variant of Ewing’s

I Z 6 Usually deep-seated, the tumour is often attached to underlying periosteum. Glycogen may be present but tends to be sparse and secondary cells are usually lacking. There is no distinct male predilection as in conventional Ewing’s sarcoma and patients tend to be older, with a median age of twenty years. Common sites include the chest wall, paravertebral regions and lower extremities and lung and bone are the usual metastatic sites. Preliminary data suggest that there is a favourable response to treatment.lZ6 The paravertebral ‘round- cell’ tumours in children reported by Tefft et ~ 1 . ’ ~ ~ are now known to belong to this group. The possibility that this form of Ewing’s sarcoma is a manifestation of an underlying undetected primary osseous Ewing’s sarcoma has been raised. 128

Fig. 25 -Frozen scction preparation of Ewing’s sarcoma showing pronounced clumped cytoplasmic positivity with PAS reaction. PAS. x 502

20 S. VARIEND

Fig. 26-Ewing’s sarcoma showing reticulin fibres limited to connective tissue and vessels. with little tendency for fibres to encompass individual tumour cells. Gomori’s reticulin, x 502

Large cell variant of Ewing’s sarcoma

The existence of an uncommon atypical or large cell variant of Ewing’s sarcoma is also recognized.’29. 130 Compared with conventional Ewing’s sarcoma, there is no difference in anatomical location or age distribution and the male preponderance is retained. Apart from the increased nuclear size (being about twice that of conventional Ewing’s sarcoma), the cells also show a greater degree of nuclear irregularity as well as increased mitotic activity. Cytoplasmic glycogen and small, dark, secondary cells are seen and nucleoli are often conspicuous. This variant of Ewing’s sarcoma, in particular, may be confused with large-cell lymphoma or the small cell type of osteosarcoma. It is claimed that similar histology may be induced in conventional Ewing’s sarcoma by radiotherapy or ~hemothe rapy . ’~~ A large cell variant is also recognized to occur in association with the soft tissue form of Ewing’s sarcoma.Iz6

Ultrastructure

Electron microscopy has proved useful in diagnosis and also in characterizing the nature of Ewing’s sarcoma.117 ‘ *c) ,131 Cells appear ovoid to polygonal in shape and the nuclear outlines are usu-

ally smooth with one or two conspicuous nucleoli. Chromatin is generally finely dispersed but marginated along the nuclear membrane. The paucity of cytoplasm is confirmed, with organelles represented by only a few mitochondria and profiles of rough endoplasmic reticulum. By contrast, free ribosomes and polyribosomes are fairly numerous.

Diagnostically, the presence of aggregated glycogen is almost certainly the most important feature.117*”8 Material prepared from paraffin blocks due to prior dissolution in formalin may not, however, reveal glycogen. Junction complexes are sparse but occur in a large proportion of tumours while intracytoplasmic microfilaments and Golgi apparatus are sometimes seen. The occasional presence of dense bodies (probably lysosomes) and lipid droplets raises the possibility that the tumour is of histiocytic lineage.54 Attention has been drawn to similarities in the ultrastructure between alveolar rhabdomyosarcoma and Ewing’s sarcoma.84 Con- densed chromatin with inconspicuous nucleoli and vacuolated cytoplasmic processes extending between other (principal) cells characterize the secondary cell. The ultrastructure of the soft tissue variant of Ewing’s sarcoma and conventional osseous Ewing’s sarcoma has been reported as being similar.132-134


CONCLUSION

Microscopic assessment of small cell tumours must take account of a full clinical history, results of special investigations such as x-rays, and the measurement of biochemical markers, all of which require a close collaboration with the clinical oncologist. Problematic histology, as determined by routine methods, should be always anticipated and measures taken so that tissue samples are set aside provisionally for special procedures. Those involved in the removal of surgical material from children with suspected neoplasms must ensure that samples reach the laboratory in a fresh condition to permit appropriate sampling and preparation for enzyme histochemistry, immunochemistry, electron microscopy and suitable imprints.

Awareness of the prototypic histology, the spectrum of variability and the usual biological behaviour of a given tumour adds significantly towards its accurate histological classification that helps, subsequently, to ensure that the most appropriate treatment programme is instituted. Conversely, the continued appraisal of therapy pro- tocols is dependent t o a great extent on accurate diagnosis.

In view of the inevitable impingement on the paediatric age range of some undifferentiated tumours commonly encountered in adults, this review cannot claim to be entirely comprehensive. Those tumours commonly encountered in children, however, have been discussed. There will remain, nevertheless, a minority of tumours with a small cell pattern that, despite the full application of current methodology, will defy diagnostic classification. Their histogenetic categorization will have to await further progrcss.

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