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in Diagnosis of Soft Tumours Cyril Fisher

To cite this version:

Cyril Fisher. Immunohistochemistry in Diagnosis of Tumours. Histopathology, Wiley, 2010, 58 (7), pp.1001. ￿10.1111/j.1365-2559.2010.03707.x￿. ￿hal-00613811￿

HAL Id: hal-00613811 https://hal.archives-ouvertes.fr/hal-00613811 Submitted on 6 Aug 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Histopathology

Immunohistochemistry in Diagnosis of Soft Tissue Tumours

ForJournal: Histopathology Peer Review Manuscript ID: HISTOP-08-10-0420

Wiley - Manuscript type: Review

Date Submitted by the 01-Aug-2010 Author:

Complete List of Authors: fisher, cyril; royal marsden hospital, histopathology

Keywords: soft tissue tumours, immunohistochemistry, , diagnosis

Published on behalf of the British Division of the International Academy of Page 1 of 39 Histopathology

Immunohistochemistry in Diagnosis of Soft Tissue Tumours

For PeerCyril FisherReview Royal Marsden Hospital, London UK

Correspondence to:

Prof Cyril Fisher MD DSc FRCPath

Dept of Histopathology The Royal Marsden Hospital 203 Fulham Road London SW3 6JJ UK

Email: [email protected] Tel: +44 207 808 2631 Fax +44 207 808 2578

Running Title : Soft Tissue Tumour Immunohistochemistry

Key Words Immunohistochemistry, sarcoma, diagnosis, soft tissue tumour

1 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 2 of 39

Abstract

Immunohistochemistry in soft tissue tumours, and especially , is used to identify differentiation in the neoplastic cells. In some cases, specific antigens are expressed; however, an initial panel of is often required in order to establish the broad lineage, with a subsequent, more focussed panel to allow classification.

Immunohistochemical evaluation must be employed with the clinical picture, the morphology, and, Forwhen necessary, Peer other ancillary Review techniques such as molecular genetics and cytogenetics.

While some diagnoses are evident on morphology, many soft tissue are seen microscopically as spindle , epithelioid cell, small round cell or pleomorphic tumours which need further to be characterized. This article reviews selected applications of immunohistochemistry in diagnosis of each of the principal morphological groups, concentrating on areas of most use in daily practice.

2 Published on behalf of the British Division of the International Academy of Pathology Page 3 of 39 Histopathology

Introduction

Soft tissue tumours comprise numerous different tumour types that are classified

according to the type of mesenchymal tissue which they resemble. The main object of

immunohistochemistry in soft tissue neoplasms, and especially sarcomas, is to identify

differentiation in the neoplastic cells. This is straightforward for those tumours that

correspond to normal tissue types, but less so for those sarcomas characterized by specific chromosomalFor translocations Peer since they Review represent novel lineages. In some cases, specific antigens are expressed; however, an initial panel of antibodies is often required

in order to establish the broad lineage, with a subsequent, more focused panel to allow

precise classification.

Tumours with adipocytic or osteochondroid differentiation or vascular space

formation can often be recognized from the microscopic appearances. Many soft

tissue neoplasms, however, show spindle cell, epithelioid cell, small round cell or

pleomorphic morphology on initial examination and require further characterisation by

immunohistochemistry, electron microscopy (although this now has a limited role 1), and

genetic analysis. This article reviews selected applications of immunohistochemistry in

diagnosis in each of the principal morphological groups, concentrating on areas of most

use in daily practice. It is important to remember that immunohistochemical evaluation

must be employed with the clinical picture, the morphology, and, when necessary, other

ancillary techniques such as molecular genetics and cytogenetics.

Spindle Cell Tumours

BENIGN SPINDLE CELL NEOPLASMS

3 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 4 of 39

The differential diagnosis of the principal benign soft tissue tumours is straightforward when the lineage is obvious. Difficulty is sometimes encountered, especially in core , in assessing , the morphological criteria for which vary according to the tumour type. In specific situations, immunohistochemistry is occasionally of assistance in distinguishing benign from malignant lesions. For example, diffuse S100 positivity in a spindled nerve sheath tumour is more suggestive of schwannoma than malignant peripheralFor nerve Peer sheath tumour Review (MPNST), especially if there are portions of capsule in the . Recently, it has been suggested that a rare CD34-positive cellular fibrous can be distinguished from the commonly CD34-positive dermatofibrosarcoma by the expression of podoplanin (D2-40) in the former but not the latter. 2 Combinations of CD34 with other antibodies can be useful in selected circumstances (Table 1). -related and proliferation markers are of little use in individual cases. The proliferation index with Ki67 can be indicative of malignancy when high, but it can also be high in reactive lesions such as nodular .

INTERMEDIATE SPINDLE CELL NEOPLASMS

Fibromatosis is a non-metastasizing lesion which is a consideration in the differential diagnostic of many types of spindle cell sarcoma. The lesional cells are which are focally positive for (SMA) and occasionally for in scattered cells. can be focally positive but CD34 is negative. Nuclear immunoreactivity for beta- is found in variable numbers of nuclei in about 80% of deep fibromatoses, but only 5% of superficial ones. 3, 4 Non-specific paranuclear staining is common in other myofibroblastic lesions and must not be misinterpreted. Nuclear

4 Published on behalf of the British Division of the International Academy of Pathology Page 5 of 39 Histopathology

beta-catenin positivity can also be found in about 40% of solitary fibrous tumours,

synovial sarcomas and endometrial stromal sarcomas, as well as in hypertrophic scars. It

is absent from gastrointestinal stromal tumour, , inflammatory

myofibroblastic tumour and MPNST, which is of use in the differential diagnosis of intra-

abdominal spindle cell neoplasms. 5 Conversely, is negative for h-

caldesmon, ALK, CD34, CD117 and DOG1. Solitary fibrousFor tumour Peer is diffusely positiveReview for CD34 in 95% of cases, as well as for bcl-2, CD99 and beta-catenin.3, 6-8 Occasional cells expressing (CK),9

epithelial membrane antigen (EMA), or S100 protein can be seen, which does not alter

the diagnosis. Malignant solitary fibrous tumour, manifesting as spindle or polygonal

cell sarcoma contiguous with (or recurrent at the site of a previous) typical solitary

fibrous tumour, usually retains CD34 expression diffusely or focally.10, 11,12

Angiomatoid fibrous histiocytoma expresses desmin in about 50% of cases, but

myogenin is negative. Other antigens that are sometimes positive are h-caldesmon, SMA

(but the morphology is unlike that of smooth muscle tumours), CD99 and EMA.13 This

tumour has three translocations resulting in the fusion EWSR1-CREB1 (the

commonest), EWSR1-ATF1 , or FUS-ATF1 .14 The first two are shared with clear cell

sarcoma (see below).

MALIGNANT SPINDLE CELL NEOPLASMS

These include , malignant peripheral nerve sheath tumour,

leiomyosarcoma, myofibrosarcoma and , and spindle cell variants of

and angiosarcoma. Non-mesenchymal tumours such as sarcomatoid

5 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 6 of 39

, and follicular dendritic cell sarcoma can present in soft tissue locations and thereby enter the differential diagnosis.

Synovial sarcoma is diagnostically obvious when biphasic. The monophasic form is a spindle cell sarcoma composed of closely packed uniform cells with overlapping nuclei and scanty , while poorly differentiated synovial sarcoma has polygonal or small round cells with high proliferation index. 15 All types of synovial sarcoma exhibit epithelial differentiationFor Peer with focal positivity Review for (including CK7 and CK19) and for EMA in most cases. Diffuse expression of bcl-2 and focal positivity for

CD99, S100 protein, CD56 and calponin are also found in some, and other non-specific markers of occasional diagnostic value include beta-catenin (nuclear) 3 and calretinin

(nuclear). 16 CD34 is almost always negative in synovial sarcoma 17 which is useful in excluding solitary fibrous tumour, since the immunophenotype of these two tumours can otherwise overlap. TLE1, an derived from expression profiling studies, is emerging as a highly sensitive (nuclear) marker for synovial sarcoma of all morphologic types. 18, 19 It is also not wholly specific, since occasional examples of nerve sheath tumours can display positivity. However, its sensitivity means that it is useful in excluding the diagnosis of synovial sarcoma when the result is negative. Reduced expression of INI1 (see below) has been described in some synovial sarcomas. 20

Synovial sarcoma is characterized by a specific chromosomal translocation t(X;18)(p11.2;q11.2), which results in fusion of the SS18 (SYT ) gene from

18 with one of several variants of the SSX gene on the .21 Recently, an antibody to the SS18 has been shown to be sensitive for identification of synovial sarcoma but it is not specific and has not found general application. 22

6 Published on behalf of the British Division of the International Academy of Pathology Page 7 of 39 Histopathology

Malignant peripheral nerve sheath tumours (MPNST) have focal nuclear

positivity for S100 protein in up to 67% of cases, which correlates with ultrastructural

evidence of differentiation. 23 There are no other ‘specific’ indicators of

nerve sheath lineage (although electron microscopy can be contributory in S100 protein-

negative examples 23 ) but many MPNST are focally positive for CD34, and a variable

proportion of cases express GFAP, basic protein, CD57 and . Cytokeratins are occasionally positiveFor but thePeer presence of CK7Review or CK19 is more indicative of synovial sarcoma than of MPNST. 24 Immunoreactivity for NSE or PGP9.5 is sometimes found in

MPNST but these markers are non-specific and of no value in diagnosis. 25 EMA

positivity is occasionally seen in MPNST with Schwannian differentiation, but is most

useful in diagnosis of benign perineurioma 26 and its rare malignant counterpart,27 which

usually lack S100 protein. Another useful marker of perineurial cell differentiation is

claudin-1 which, as a -associated protein,28 has a granular distribution in

normal perineurial cells and in perineurioma.29 Claudin-1 has also been reported as

positive in low-grade fibromyxoid sarcoma 30 which can resemble perineurioma,

especially in cutaneous lesions, so that genetic analysis (see below) can be required for

diagnosis.

Leiomyosarcoma is most often morphologically distinctive, with its non-tapered

cells with cytoplasm and squared-off nuclei, arranged in cellular fascicles in

a distinctive rectilinear pattern. Immunohistochemically, desmin, SMA, muscle specific

actin (MSA), h-caldesmon, calponin and smooth muscle (SMM) are expressed in

the majority of . Some examples co-express cytokeratins (frequently

with dot pattern), EMA and S100 protein but usually only when the muscle-specific

7 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 8 of 39

antigens are present. CD99 can also be seen in a paranuclear dot pattern. CD34 expression is variable but generally negative in leiomyosarcoma, and CD117 is absent from smooth muscle tumours. A spindle cell expressing SMA but not desmin or h-caldesmon is unlikely to be a smooth muscle tumour and is more likely to be myofibroblastic.31

Myofibrosarcomas are spindle cell or pleomorphic sarcomas which display myofibroblastic differentiation.For Peer32 Low grade Reviewmyofibrosarcomas are composed of tapered cells containing ovoid nuclei and punctate nucleoli, arranged in sheets and fascicles. 33

They are positive for SMA (with a ‘tram-track’ subplasmalemmal accentuation), and usually express calponin and less commonly desmin. H-caldesmon and SMM are negative, distinguishing myofibrosarcoma from leiomyosarcoma. 34 CD34 and S100 protein are also negative; the combination of CD34 and desmin immunoreactivity is found in mammary-type myofibroblastomas, 35 but their ultrastructure is more like that of smooth muscle cells than of true myofibroblasts. 36 Inflammatory myofibroblastic tumour

(IMT) is a low-grade myofibroblastic malignancy with fasciitis-like, fascicular and sclerosing histological patterns. 37 In addition to the usual myofibroblastic markers, about half of IMT, especially those in viscera and in children, express ALK, and recent evidence suggests that lack of ALK is prognostically adverse.38 In IMT, the ALK gene is rearranged with one of six partner genes, most of which result in cytoplasmic staining on immunohistochemistry, except for ALK-RANBP2 . The latter gives a distinctive nuclear membranous distribution as RANBP2 binding are localised at the complex.39

8 Published on behalf of the British Division of the International Academy of Pathology Page 9 of 39 Histopathology

Adult fibrosarcoma is now a rare tumour which is by definition negative for all

lineage markers ( express but this is a wholly non-specific marker

which need not usually be included in diagnostic panels). However, specific subtypes of

fibrosarcoma are positive for CD34, including those arising in dermatofibrosarcoma,

about half of myxofibrosarcomas, and a smaller proportion of myxoinflammatory

fibroblastic sarcomas. 40 Some superficial fibrosarcomas that are strongly CD34 positive but lack an apparentFor dermatofibrosarcomatous Peer Review component have been shown to have the same COL1A1-PDGFRB fusion gene transcripts as dermatofibrosarcoma. 41 In a CD34-

negative fibrosarcoma, an occasional SMA-positive cell can sometimes be seen which is

attributable to focal myofibroblastic differentiation, but diffuse or multifocal SMA

staining indicates categorization as myofibrosarcoma. 32

Low grade fibromyxoid sarcoma occasionally has SMA or CD34 positivity (and,

as previously noted, can express EMA and claudin-130 ), but the diagnosis is best

confirmed by genetic demonstration of the t(7;16)(q33;p11) or FUS-CREB3L2 (or rarely

FUS-CREB3L1 ) fusion gene, 42-44 which has also been reported in some examples of

sclerosing epithelioid fibrosarcoma. 45

Spindle cell rhabdomyosarcoma of juvenile (adolescent) 46, 47 or adult type 48-50 is

diffusely positive for desmin, and at least focally so in nuclei for myogenin and MyoD1.

The latter is less sensitive, but in the rare sclerosing pseudovascular rhabdomyosarcoma,

MyoD1 is more widely expressed than myogenin. 51 It should be noted that these are

nuclear antigens and that cytoplasmic staining for either is non-specific and does not

denote rhabdomyoblastic differentiation. Myoglobin, a previously widely used

9 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 10 of 39

cytoplasmic marker for differentiation, is of low sensitivity and specificity and is no longer recommended for routine use.

Spindle cell angiosarcoma is an uncommon soft tissue sarcoma of skin or soft tissue in which diagnostic vasoformative areas can be present only focally and therefore absent from a core biopsy. The tumour can be identified by its expression of CD31, and less commonly of CD34 and other endothelial markers (see discussion of epithelioid endothelial neoplasmsFor below). Peer CD117 Review positivity has also been reported in angiosarcoma 52 and in Kaposi sarcoma (KS). The latter, however, demonstrates highly specific immunoreactivity for HHV8 (KSHV) in the nuclei of its spindled and endothelial cells, which is not found in other endothelial neoplasms. 53 Podoplanin (D2-40) is a lymphatic endothelial marker that has a membranous staining pattern in Kaposi sarcoma, but also in some angiosarcomas and gastrointestinal stromal tumours (which also display

CD34 and CD117, as in angiosarcoma).54 Antibodies to vascular endothelial growth factor and its (such as VEGF-C and VEGFR-3) are also immunoreactive in

KS. 55, 56

Follicular dendritic cell sarcoma (FDCS), which sometimes arises in soft tissue sites,57-59 is characterised by fascicles and whorls of cells in which the nuclei typically display prominent membranes and a speckled chromatin pattern. Immunoreactivity for

CD21, CD35 (the two are more sensitive when used together as a cocktail 60 ) and CD23 is diagnostic, and other markers reportedly helpful in identifying FDCS include , clusterin 61 and D2-40.62 Many examples are also positive for S100 protein and EMA. 63

Sarcomatoid carcinoma can express cytokeratins focally, especially those of high molecular weight, but can also be negative. 64 The diagnosis can be facilitated by finding

10 Published on behalf of the British Division of the International Academy of Pathology Page 11 of 39 Histopathology

areas of epithelial differentiation or surface dysplasia, or a nested reticulin pattern.

Spindle cell carcinoma can also express SMA, but desmin, h-caldesmon, CD34 and S100

protein are absent. SMA-positive/CK-negative , which is usually

a pleomorphic neoplasm, can be difficult to distinguish from pleomorphic

myofibrosarcoma without knowledge of the history and the location of the tumour.

Gastrointestinal stromal tumours (GIST) can arise not only in the but also in For extra-gastrointestinal Peer locations, Review especially omentum, mesentery and retroperitoneum. The two predominant patterns are spindled or epithelioid, which have a

similar immunophenotype. GIST typically express CD117, the product of the KIT gene,

which encodes a receptor tyrosine kinase, and which in GIST shows a variety of

prognosis-related . 65-67 A small number of GIST are CD117-negative, and

some of these have mutations in the gene which encodes -derived growth factor

receptor A (PDGFRA). 67 These are often epithelioid gastric GIST. The antibody DOG1,

identified by profiling, has high sensitivity and specificity for GIST and

can identify most KIT-negative tumours. 68, 69 Other antigens found in GIST include

CD34 in 50-100% of tumours (varying with site 70 ), SMA (varying inversely with CD34

expression), h-caldesmon, bcl-2 and, less commonly, desmin or S100 protein. Beta-

catenin is negative in nuclei, in contrast to fibromatosis. 5

Epithelioid and clear cell tumours

Epithelioid soft tissue neoplasms include , and epithelioid variants of

MPNST, leiomyosarcoma, and fibrosarcoma, as well as clear sarcoma, alveolar soft part

sarcoma, PEComa (Table 2), and melanoma and carcinoma when presenting in soft

tissue locations.

11 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 12 of 39

Epithelioid sarcoma (EpS), of both classical and proximal types, is positive for the epithelial markers cytokeratins and EMA and, in about 50% of cases for CD34. 71

This is particularly useful in making the distinction from primary or metastatic , which are CD34 negative. In addition, CK5/6, a useful marker for carcinomas especially those with squamous differentiation, is detectable in only a small proportion of epithelioid sarcomas.72 EpS generally lacks desmin, S100 protein and FLI- 1, 73 which help For in diagnosis Peer from rhabdomyosarcoma, Review melanoma, and epithelioid angiosarcoma respectively. Antibodies to INI1 are negative in about 90% of EpS, in malignant rhabdoid tumours (MRT), in about half the cases of epithelioid MPNST, and in some extraskeletal myxoid with rhabdoid cytomorphology.74-76 This relates to the location of the SMARC/INI gene on chromosome 22q, which is mutated or deleted in MRT and EpS.74 Absence of INI1 is extremely useful for diagnosis of these tumours since all other epithelioid or polygonal cell tumours in the differential diagnosis display nuclear positivity for this marker. MRT can be distinguished from EpS by absence of CD34, although clinical data are required to distinguish MRT from CD34- negative EpS.

Epithelioid MPNST differs from its spindle cell counterpart in displaying diffuse

S100 protein expression in most cases.77 It differs from melanoma in having more uniform cytological features, frequent association with a typical spindle cell nerve sheath component, and absence of melanoma-specific antigens. INI-negative cases should not be misdiagnosed as epithelioid sarcoma, or INI-positive cases as epithelioid angiosarcoma, since both of these tumour types are S100 protein negative.

12 Published on behalf of the British Division of the International Academy of Pathology Page 13 of 39 Histopathology

Epithelioid endothelial neoplasms manifest varying combinations of vascular

endothelial cell markers. These include CD34, CD31, FLI-1 (in nuclei), FVIIIRAg,

and CD117. In epithelioid angiosarcoma and malignant variants of

epithelioid haemangioendothelioma these antigens are selectively expressed so that not

all are detectable by immunohistochemistry. CD31 and FVIIIRAg are the most specific,

although the latter is not very sensitive. These tumours show diffuse nuclear immunoreactivity For for INI1, 76 Peer and D2-40 isReview positive in some cases.54 In addition, epithelioid angiosarcoma is frequently cytokeratin-positive, while usually lacking

EMA. 78, 79 Epithelioid sarcoma, especially the proximal variant, can be morphologically

similar to epithelioid angiosarcoma and can also co-express CD34 and cytokeratins, but

lacks CD31, FVIIIRAg, FLI-1, and INI1. Epithelioid sarcoma-like

haemangioendothelioma is a very rare low-grade spindle cell neoplasm that displays

positivity for cytokeratins, CD31, and FL1-1, but lacks CD34 (Table 1).80, 81

PEComa is the term used for a group of clinically disparate tumours composed of

supposed perivascular epithelioid cells – spindle or polygonal cells with clear or granular

cytoplasm and uniform nuclei, which are arranged in nests around sinusoidal vessels.82, 83

Members of the PEComa ‘family’ include , ,

clear cell ‘sugar’ tumour of and other sites, and clear cell myomelanocytic tumour.

PEComas co-express SMA and the melanocytic antigens HMB-45, melan-A, and micro-

ophthalmia factor (MITF). It has become recognized that PEComas can

also focally express S100 protein, desmin and h-caldesmon as well as TFE3 in some

cases. 84 Most PEComas are benign but malignant examples are increasingly reported.84, 85

The principal differential diagnosis (Table 2) is with clear sarcoma of and

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aponeuroses which also expresses melanoma-specific antigens, but which has diffuse

S100 protein positivity, lacks SMA and h-caldesmon, and is characterized by a specific translocation t(12;22)(q13;q12) with fusion of EWS and ATF-1 genes. 86 The morphologically similar of the gastrointestinal tract with osteoclast- like giant cells is also S100 protein positive but lacks melan-A and HMB45 expression, and usually has t(2;22)(q33;q12) with EWS-CREB1 fusion. 87 However, examples of both fusions have beenFor described Peer at both sites. Review88 The same fusions are also found in angiomatoid fibrous histiocytoma, 89 representing one of several examples of promiscuity among gene fusions. 90

Alveolar soft part sarcoma has long been an enigmatic tumour in which the occasional report of immunoreactivity for desmin and, for a time, MyoD1, led to the assumption that the tumour showed skeletal muscle differentiation. This tumour, however, is now known to be a translocation-associated sarcoma characterized by t(X;17)(p11;q25) with fusion of TFE3 and ASPL genes.91 Antibodies to TFE3 are available which demonstrate nuclear positivity in alveolar soft part sarcoma; similar staining is also found in some translocation-associated carcinomas of , in some

PEComas and in examples of granular cell tumour.92

Pleomorphic tumours

Pleomorphic sarcomas include (pleomorphic and dedifferentiated), rhabdomyosarcoma, MPNST, leiomyosarcoma, myofibrosarcoma, and undifferentiated pleomorphic sarcoma (also known as pleomorphic malignant fibrous histiocytoma or

14 Published on behalf of the British Division of the International Academy of Pathology Page 15 of 39 Histopathology

MFH). Undifferentiated or sarcomatoid carcinoma, melanoma and rarely lymphoma can

also have similar microscopic appearances.

Pleomorphic neoplasms are composed predominantly of pleomorphic spindle,

polygonal and giant cells with mitoses, necrosis and . In addition the

specific subtypes have foci of differentiation perceived either morphologically

(lipoblasts) or immunohistochemically (skeletal, smooth muscle, melanocytic or epithelial antigens).For In many Peercases without apparentReview lineage, the pleomorphic cells have ultrastructural features of with a variable proportion of cells showing

myofibroblastic differentiation. 93 Thus, MFH and undifferentiated areas of other

pleomorphic sarcomas can display focal SMA positivity, with a characteristic

subplasmalemmal linear distribution imparting a ‘tram-track’ appearance. 94 Desmin

positivity is occasionally seen but h-caldesmon is absent. It is important to identify

pleomorphic sarcomas with any type of myogenic differentiation at

immunohistochemical level since, counter-intuitively, they have a worse prognosis than

undifferentiated pleomorphic sarcomas. 95, 96 Pleomorphic rhabdomyosarcoma usually

has diffuse desmin positivity and focal nuclear staining for myogenin. MyoD1 is a less

sensitive marker except in sclerosing pseudovascular rhabdomyosarcoma.51, 97 In

pleomorphic leiomyosarcoma , typical fascicular areas occupying <25% of the

pleomorphic tumour are usually required for the diagnosis.98 However,

immunohistochemical evidence of smooth muscle differentiation (desmin, SMA and h-

caldesmon) without corresponding morphological features is associated with a worse

prognosis.95, 96 MFH-like undifferentiated sarcomas with widespread SMA positivity can

be termed pleomorphic myofibrosarcomas; the distinction is important since

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myofibroblastic differentiation, although less well-studied than myoid differentiation, also appears prognostically unfavorable. 94

Dedifferentiated liposarcoma , like well differentiated liposarcoma, has MDM2 and CDK4 amplification, detectable by fluorescence in situ hybridization (FISH) and also by immunohistochemistry.99 Most retroperitoneal (and some extremity 100 ) undifferentiated pleomorphic sarcomas (MFH) and myxofibrosarcomas express these markers (in nuclei)For and are nowPeer considered Review to represent dedifferentiated even when no well-differentiated component remains. 100-103 In addition, dedifferentiated liposarcoma can express actin, desmin and CD34 focally in the pleomorphic areas,101 which should not be misinterpreted as leiomyosarcoma.

Sarcomatoid carcinoma can appear as a pleomorphic neoplasm. This usually expresses cytokeratins in a multifocal or diffuse distribution that is best demonstrated with a broad spectrum antibody or ‘cytokeratin cocktail’. Even very focal cytokeratin expression should prompt consideration of a primary carcinoma, and it can be accepted as evidence of epithelial differentiation in an organ-based (visceral) pleomorphic neoplasm.

It should be remembered, however, that some undifferentiated pleomorphic sarcomas in soft tissue have (aberrant) expression of cytokeratin, usually in a few scattered cells. 104,

105 Also, as previously noted, spindled carcinomas can acquire myofibroblastic differentiation and thereby be focally positive for SMA (but not desmin or h-caldesmon).

As always, the final diagnosis must summate all data including clinical and radiological findings.

Small round cell tumours

16 Published on behalf of the British Division of the International Academy of Pathology Page 17 of 39 Histopathology

This group of soft tissue neoplasms includes Ewing sarcoma/primitive

neuroectodermal tumour (ES), desmoplastic small round cell tumour (DSRCT), alveolar

rhabdomyosarcoma (ARMS), (NB), and poorly differentiated synovial

sarcoma (PDSS). Most of these occur predominantly in childhood and adolescence, but

are also seen sporadically in adults. 106 Several of these sarcoma subtypes have specific,

translocations and consequent fusion genes that allow precise diagnosis, and in some instances provide For prognostic Peer information, usingReview FISH or PCR-based methods. 90, 107 A molecular pathology service is not always readily available, but the immunophenotypes

of these small round cell sarcomas differ sufficiently to enable diagnosis in many cases.

Lymphomas, especially of lymphoblastic type, must also be considered in the differential

diagnosis, and TdT should be part of the diagnostic panel in the appropriate clinical

setting. Similarly leukaemic deposits, and small cell carcinomas might need exclusion by

immunostaining for myeloid markers or cytokeratins, neuroendocrine antigens and TTF1

respectively.

Ewing sarcoma is a translocation-associated tumour, which can arise in or

extraskeletal sites; examples with evidence of neural differentiation are termed PNET. 90

The large majority of ES display diffuse membranous staining for CD99, and some

express proteins and NSE. About 70% show nuclear immunoreactivity for

FLI-1. 108 A small number are also positive for S100 protein or display dot positivity with

broad spectrum anticytokeratin antibodies and membranous staining for EMA. CD56 is

usually negative, unlike in small cell neuroendocrine carcinoma and PDSS.109 The latter

also expresses TLE1, but some cases have a similar immunophenotype to ES and

although there are subtle morphological differences (ES tends to be more uniform and

17 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 18 of 39

lacks intercellular reticulin), genetic analysis may be required to make the diagnosis. The morphologic spectrum of ES is expanding due to increased recognition of atypical variants aided, in part, by molecular studies.110, 111 Although there is a wide range of available antibodies at least three of a panel composed of CD99, FLI-1, HNK1, and -1 reportedly show immunoreactivity in all cases with caveolin-1 being positive in CD99-negative cases. 111 DesmoplasticFor small roundPeer cell tumour Review was originally described in the abdomen and pelvis in adolescent males, and is now recognized in other locations. It shows presumed primitive mesothelial differentiation, and has a characteristic t(11;22)(p13;q12) rearrangement with formation of an EWS-WT1 fusion gene. Immunohistochemically,

DSRCT shows nuclear staining with antibodies to the C-terminal end of WT1 as well as multilineage differentiation with dot positivity for desmin, cytokeratins (CK20 and

CK5/6 are negative), and neural markers including , , , and CD56. 112, 113 However, not all lineage markers are expressed in every case.114 DSRCT is usually negative for CD99 but 10-20% (and especially those with variant translocations) can be positive which does not therefore exclude the diagnosis. 115, 116

Alveolar rhabdomyosarcoma , as with other rhabdomyosarcoma subtypes, has diffuse cytoplasmic staining for desmin. Myogenin is expressed in a majority of tumour cell nuclei and is more sensitive than MyoD1. 117 CD56 is also positive as in other types of rhabdomyosarcoma. 70-90% of ARMS have either t(2;13)(q35;q14) creating a PAX3-

FOXO1 fusion gene or t(1;13)(p36;q14) with resulting PAX7-FOXO1 fusion. 118

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Immunohistochemistry with an antibody to PAX5 can detect all translocation-positive

cases of either type.119

Neuroblastoma can be recognized not only by its clinical context (the vast

majority are in patients <4 years old and arise in adrenal or other sympathetic nervous

system locations), but also by its expression of NB84, NSE, CD56 and neurofilament

proteins. 120 Antibodies to these markers, which can be used as a panel, are particularly useful in detectionFor of residual Peer Review in bone marrow specimens taken after . Neuroblastoma is negative for cytokeratins, desmin and CD99, which can

be particularly helpful in diagnosis of rare examples in adults. 121

Adipose tumours

The distinction between and atypical lipomatous tumour (ALT)/well

differentiated liposarcoma can be difficult since it depends on focal morphologic

subtleties which are not always represented in the samples taken. The diagnosis is

facilitated by the demonstration of immunohistochemical positivity for products of the

murine double minute type 2 ( MDM2) and cyclin-dependent kinase 4 ( CDK4 ) genes

which are overexpressed in ALT as a result of amplification in the 12q14-15

chromosomal region. All ALT are immunoreactive in nuclei for MDM2 (often focally)

and 91% for CDK4 (more diffusely).99 These have application not only in the differential

diagnosis of lipoma and ALT, but also in determining whether the fatty tissue at the

margin of excision of ALT is neoplastic or not. As mentioned above, in dedifferentiated

liposarcoma (DDL), which is genetically related to ALT, CDK4 and MDM2 are similarly

overexpressed.102, 122 Their specificity in DDL is reduced since a small proportion of

19 Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 20 of 39

MPNST, myxofibrosarcomas and embryonal are immunoreactive with both 99 but, apart from morphological considerations, the diagnosis of DDL is supported by positivity for these two markers in the differentiated adipose tissue adjacent to the pleomorphic neoplasm.

Conclusion Existing antibody Forpanels, when Peer correctly selected, Review have a major role in diagnosis of soft tissue sarcomas. Diagnostically useful new antibodies, such as DOG1 and TLE1, are emerging from gene profiling studies, and in addition to the usual reagents, antibodies to fusion gene products might theoretically be of value in detecting translocation sarcomas.

For example, antibodies raised against TLS/EWS-CHOP chimeric oncoproteins have shown high specificity and sensitivity for myxoid and round cell liposarcoma. 123 These approaches can be expected to lead to improved diagnosis of soft tissue tumours and perhaps to more efficient use of immunohistochemistry with limited and therefore less expensive panels.

Acknowledgment

I acknowledge NHS funding to the NIHR Biomedical Research Centre .

20 Published on behalf of the British Division of the International Academy of Pathology Page 21 of 39 Histopathology

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36 Published on behalf of the British Division of the International Academy of Pathology Page 37 of 39 Histopathology

Table 1. CD34 and other markers in selected spindle and epithelioid soft tissue tumours

CD34 CK SMA S100pr Desmin Other diagnostic markers

Dermatofibrosarcoma + - - + (Bednar) − For Peer Review Ectopic hamartomatous thymoma + + − − −

Epithelioid angiosarcoma + + − − − CD31, INI1

Epithelioid sarcoma + + ± − − INI1 negative

Gastrointestinal stromal tumour + − + ± CD117, DOG1, h-caldesmon

Mammary type myofibroblastoma + − ± − +

Neurofibroma + − − + −

Perineurioma + − − − − EMA, claudin-1

Peripheral nerve sheath tumour, malignant + ± − + focal + (Triton)

Schwannoma + ± − + diffuse − GFAP

Solitary fibrous tumour + − − − − Bcl-2, CD99, CD56

Epithelioid malignant peripheral nerve sheath tumor − − − + − INI1 (50% negative)

Epithelioid sarcoma-like haemangioendothelioma − + − − − CD31+, FLI1+

Fibromatosis − − + ± ± Beta-catenin

Published on behalf of the British Division of the International Academy of Pathology Histopathology Page 38 of 39

Inflammatory myofibroblastic tumour − ± + − − ALK (50%)

Myofibrosarcoma − − + − ± Calponin, h-caldesmon negative

Retroperitoneal fibrosis − − + − − IgG4/IgG ratio

Smooth muscle tumours − ± + + h-caldesmon, SMM For Peer Review Spindle cell carcinoma − + + − − EMA, CK5/6, CK34betaE12

Synovial sarcoma − + − ± − TLE1, CD99, CD56

± = in some cases, CK = cytokeratin. EMA = epithelial membrane antigen, GFAP = glial fibrillary acidic protein, SMM = smooth muscle myosin

Published on behalf of the British Division of the International Academy of Pathology Page 39 of 39 Histopathology

Table 2. Differential diagnosis of soft tissue tumours with clear cells

S100 protein HMB45 Melan-A SMA Desmin TFE3 Other diagnostic findings

Clear cell sarcoma (soft tissue) + (diffuse) + + - - - t(12;22)(q13;q12), EWSR1-ATF1

Clear cell sarcoma (GIT) +For (diffuse) Peer - Review - - - - t (2;22)(q33;q12), EWSR1-CREB1

PEComa ± (focal) + + + ± ±

Paraganglioma - (except sustentacular - - - - - CD56+, NF+, CG+ cells)

Alveolar soft part sarcoma - - - - ± + t(X;17)(p11;q25) , ASPL-TFE3

± = in some cases , GIT = gastrointestinal tract, SMA = smooth muscle actin, NF = neurofilament, CG = chromogranin,

Published on behalf of the British Division of the International Academy of Pathology