JOURNAL OF PATHOLOGY, VOL. 162: 29-33 (1990)

ALPHA-SMOOTH MUSCLE ACTIN AS A MARKER FOR SOFT TISSUE TUMOURS:

A COMPARISON WITH DESMIN HUGH JONES, PHILLIP V. STEART, CLAIR E. H. D U BOULAY AND WILLIAM R. ROCHE

Department of Pathology, Southampton General Hospital and University of Southampton, Southampton SO9 4 X Y , u. K.

Received 12 February 1990 Accepted2 April 1990

SUMMARY The immunoreactivity of a range of vascular and non-vascular smooth muscle tumours, rhabdomyosarcomas, and

non-myoid lesions has been examined with the use of a monoclonal antibody to smooth muscle-specific actin and the muscle intermediate filament, desmin. In all cases of smooth muscle-derived tumours, the alpha-actin antibody yielded superior results. Staining of the myofibroblasts of fibromatoses was also seen. In contrast to desmin, immunoreactivity was not exhibited by rhabdomyosarcomas. We propose that this monoclonal antibody to alpha-smooth muscle actin is a useful addition to the panel of reagents used for the characterization of soft tissue proliferations and tumours. The technical aspects of the application of this monoclonal antibody to immunohistochemistry are discussed.

KEY WORDS-Actin, desmin, sarcoma, immunohistochemistry.

INTRODUCTION

The role of immunohistochemistry in the differ- ential diagnosis of soft tissue tumours is now well established.' These lesions exhibit a wide range of morphological appearances and the classification of tumours by conventional light microscopy alone is often incomplete and inadequate. Immunohisto- chemistry also has an important role in the develop- ment of our understanding of the large group of tumours whose histogenesis remains unclear, despite their distinctive histological appearances.'

Smooth muscle tumours represent one of the largest diagnostic categories of soft tissue neo- plasms, and malignant smooth muscle tumours rep- resent 7 per cent of ali sarcomas.2 Commercially available monoclonal antibodies to desmin are commonly used to aid the identification of these tumours in routine histopathological practice. Although the presence of desmin has been described in skeletal muscle, them ocardium, and the smooth muscle of the viscera:>'wherein it acts as a cyto-

Addressee for correspondence: Dr W. R. Roche, Department of Pathology, University of Southampton, Level E, South Block, Southampton General Hospital, Southampton SO9 4XY, U.K.

skeletal support for contractile myofilament~,~ its value in the diagnosis of smooth muscle tumours is limited. Our experience is in keeping with that of Enzinger, who has described the absence of immuno- reactivity in the majority of leiomyosarcomas of the soft tissue.6 Furthermore, many laboratories have experienced difficulty in the use of antibodies to desmin and its staining properties o n paraffin- embedded material are not consistent.6

Actins are abundant globular intracellular pro- teins which polymerize into filaments which are essential for cytoskeletal structure, cell motility, and smooth muscle c~nt rac t jon .~ In mammalian cells there are at least six isoforms of actin, as character- ized by amino acid sequen~ing .~ .~ Although these actin isoforms show an overall sequence homology which exceeds 90 per cent, there is diversity in the 18 N-terminal residues."," The N-terminal region is a major antigenic component of the molecule and as amino acid homology is restricted to 50-60 per cent in this region, isoform-specific antibodies may be raised.

Although the relative distribution of actin iso- forms in smooth muscle differs between organs and stages of de~eloprnent , '~"~ two specific actin iso- forms, alpha-smooth muscle actin (ASMA) and

0022-341 7/90/09002945 $05.00 0 1990 by John Wiley & Sons, Ltd.

30 H. JONES ET AL.

Table I-Immunoreactivity of soft tissue lesions

Desmin ASMA Initial diagnosis + + + ++ + Negative + + + + + + Negative

Leiom yoma Cutaneous Uterine

Leiom yosarcoma Rhabdomyosarcoma Haemangiopericytorna Glomus tumour MFH* Fibromatosis

1 3 2 3 -

- 2 -

- 1 2

I 3 5 I 5 5

10 1 5 1

-

-

-

*Four of these cases were assigned to other diagnostic categories

gamma-smooth muscle actin, predominate. We have examined the pattern of immunoreactivity of smooth muscle tumours to a commercially available monoclonal antibody produced to a synthetic amino end terminal decapeptide of the alpha- smooth muscle specific actin i s ~ f o r m . ' ~

MATERIALS AND METHODS

The soft tissue tumours used in this study were obtained from consecutive surgical histology material at Southampton General Hospital. The tumours were selected by the original diagnosis, which was reviewed by a histopathologist with a special interest in soft tissue tumours (C du B). Ten leiomyosarcomas, eight rhabdomyosarcomas (four embryonal and four alveolar), 12 malignant fibrous histiocytomas, six leiomyomas (three cutaneous and three uterine), seven haemangiopericytomas, five glomus tumours, and five fibromatoses had been previously diagnosed using light microscopy with tinctorial and immunoperoxidase staining and, in some cases, by electron microscopy. Blocks which had been fixed in 10 per cent neutral buffered forma- lin and embedded in paraffin were retrieved from the files. Sections were cut at 5pm, dewaxed, and the sections for the demonstration of ASMA were incubated for 10 min in 0.1 per cent trypsin in 0.1 per cent calcium chloride. Sections were incubated with monoclonal antibodies to desmin (Bio Nuclear Services, Reading, U.K., code MDE), at a dilution of 1:20 for 30 min at room temperature, or to ASMA (Sigma Chemical Co., Poole, U.K., clone 1A4), at 1:8000 dilution at 4°C for 16 h. Immuno- reactivity was demonstrated by the avidin-biotin-

peroxidase technique (Dako, High Wycombe, U .K .). l 5

The sections were examined independently by two observers (HJ and WRR), without knowledge of the histological diagnosis. There was good con- cordance between the observers, and an agreed grade was assigned on a semi-quantitative + 1, + 2, + 3 basis.

RESULTS

The monoclonal antibody to ASMA yielded strong immunoreactivity even at dilutions of 1 in 10 000. This was confined to cell cytoplasm. Reac- tivity was found to be dependent on fixation, the strongest reaction being found in small biopsies and in areas which had undergone good fixation, such as the peripheral portion of tumours in large resection specimens. Staining of the cytoplasm of plasma cells was also seen. In all cases which exhibited immuno- reactivity to both antibodies, the strength of the reaction product with ASMA exceeded that of desmin.

The results of the comparison of these two anti- bodies are shown in Table I. All six leiomyomas and three of ten leiomyosarcomas showed positivity for desmin. In contrast, all the smooth muscle lesions were positive for ASMA. In tumours which showed some epithelioid differentiation, the actin immuno- staining was confined to the spindle cell component. None of the eight rhabdomyosarcomas exhibited positivity for ASMA, while three cases showed focal desmin positivity.

Neither the haemangiopericytomas nor the glomus tumours were stained by the monoclonal

a-SMOOTH MUSCLE ACTIN IMMUNOHISTOCHEMISTRY 31

Fig. 1-Immunoperoxidase demonstration of ASMA. (A) Cutaneous leiomyoma showing strong staining of the tumour cell cytoplasm and of the tunica media of a small vessel. (B) Leiomyosarcoma showing a fibrillar cytoplasmic staining pattern. (C) Haemangiopericytoma-staining is confined to the smooth muscle of a tumour blood vessel. (D) Glomus tumour showing distinct perinuclear staining of the tumour cells. (E) Fibromatosis showing variable staining of the proliferatingcells, which is weaker than that exhibited by an adjacent blood vessel. (F) Malignant fibrous histiocytoma4espite intense staining of the smooth muscle of a blood vessel, the tumour cells and vascular endothelium are negative

32 H. JONES ET AL.

antibody to desmin. ASMA was demonstrated strongly and consistently in the perivascular cells of the glomus tumours, while staining of the hae- mangiopericytomas was either weak or absent (Fig. 1). Review of the tumours initially diagnosed as malignant fibrous histiocytoma without knowledge of the ASMA results led to the reclassification of four cases: two as leiomyosarcoma, one of which revealed focal desmin positivity and both of which were positive for ASMA; one as rhabdomyosar- coma in which desmin was positive and ASMA negative; and one case as malignant Schwannoma which was negative for both desmin and ASMA. Seven out of the remaining eight cases of malignant fibrous histiocytoma were negative for ASMA and all were negative for desmin. One case which had the histological features of a malignant fibrous histio- cytoma was positive for ASMA, reflecting the heterogeneous nature of the tumours included under this rubric. Among the fibromatoses, desmin was negative but all cases were positive for ASMA, although the strength of the immunoreactivity was variable and inconsistent (Fig. 1).

DISCUSSION

The results obtained herein for desmin immuno- reactivity are lar ely in agreement with pre- vious authors,6.'631B although the proportion of leiomyosarcomas which exhibited positivity was less than that reported by some authors." Haeman- giopericytomas and glomus tumours have been reported consistently as being negative for des- min. 17.19 While leiomyomas are generally regarded as being desmin positive,I7 a variable proportion of leiom yosarcomas exhibit immunoreactivity for this intermediate filament. The desmin reactivity of rhabdomyosarcomas is also variable and is thought to reflect the degree of differentiation of these tumours.20

The results obtained with the monoclonal anti- body to ASMA were in marked contrast to those obtained with desmin. In all sections, the antibody showed strong positivity in the tunica media of blood vessels. This provides a consistent positive internal control. Both benign and malignant smooth muscle tumours demonstrated immunoreactivity, although this was confined to spindle cell areas in leiomyosar- comas showing epithelioid differentiation. The diag- nostic power of this antibody was further enhanced by the consistent negativity of rhabdomyosarcomas.

Among the vascular tumours, the uniform strong positivity and characteristic cytoplasmic staining of

glomus tumours readily allowed them to be dis- tinguished from haemangiopericytomas. This is in contrast to the results obtained with non-isotype specific actin antibodies which do not discriminate between these tumours.' Differentiation between these two neoplasms may sometimes be difficult and application of the ASMA monoclonal antibody may assist in differential diagnosis. This clear difference in the expression of ASMA may reflect differences in the histogenesis of these neoplasms.

The immunoreactivity of fibromatoses is in keep- ing with a previous report2' and probably reflects the myofibroblastic nature of the proliferating cells in these lesions. The characteristic appearances of these lesions are such that this phenomenon should not create a problem in excision specimens, although caution may be required in the interpret- ation of immunochemical results obtained with the ASMA antibody in small biopsy specimens. Our reclassification of a considerable proportion of the lesions diagnosed as MFH is in keeping with the contemporary experience with this tumour. The application of more powerful diagnostic reagents and the greater experience of histopathologists in this area are gradually reducing the diagnostic incidence of these tumours."

Although the group which produced this mono- clonal antibody to ASMA have described its reac- tivity in a small range of smooth muscle tumours22 and have used it as part of a panel for the character- ization of the differentiation of rhabdomyosar- comas,16 they have not advocated its application to the diagnostic problem of smooth muscle tumours. This may be because their results were based on the less satisfactory method of immunofluorescence on paraffin-embedded tissue. We propose this new antibody as a reliable and specific addition to the routine diagnostic panel of monoclonal antibodies required for the characterization of tumours arising in the soft tissues.

ACKNOWLEDGEMENTS

The authors thank Miss S. Harris for printing the photomicrographs and Miss M. D. Harris for her secretarial assistance.

REFERENCES

1. du Boulay CEH. Irnrnunohistochernistry of soft tissue turnours: a

2. Russell WO, Cohen J , Enzinger FM, eta!. A clinical and pdtho\ogica\ review. J Parho! 1985; 146: 77-94.

staging system for soft tissue sarcomas. Cancer 1977; 4 0 1562-1 570.

a-SMOOTH MUSCLE ACTIN IMMUNOHISTOCHEMISTRY 33

3. Bennett GS, Fellini SA, Croop JM, Otto J J , Bryan J, Holtzer H. Differences among 100A-filament subunits from different cell types. Proc Natl AcadSci U S A 1978; 7 5 4364-4368.

4. Lazarides E, Balzer DR. Specificity of desmin to avian and mammalian muscle cells. Cell 1978; 1 4 429-438.

5. Tokuyasu K, Dutton A, Singer S. lmmunoelectron microscopic studies of desmin (skeletin) localisation and intermediate filament organization in chicken skeletal muscle. J Cell Biol 1983; 96: 1727-1742.

6. Enzinger FM, Weiss SW. Soft Tissue Tumors. 2nd ed. St Louis: C V Mosby, 1988: 414.

7. Pollard TD, Cooper JA. Actin and actin binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Eiochem 1986; 5 5 987- 1035.

8. Garrels JI , Gibson W. Identification and cbaracterisation of multiple forms of actin. Cell 1976; 9 793-805.

9. Sorti RV, Rich A. Chick cytoplasmic actin and muscle actin have different structural genes. Proc Narl Acad Sci USA 1976; 73: 2346-2350.

10. Benyamin Y, Roustan C, Boyer M. Anti-actin antibodies. Chemical modification allows the selective production of antibodies to the N-terminal region. JlmmunolMerhods 1986; 86 21-29.

I I . Bulinski JC, Kumar S , Titani K, Hauschka SD. Peptide antibody specificity for the amino terminus of skeletal muscle a actin. Proc Nafl AcadSci USA 1983;80 1506-1510.

12. Gabbiani GE, Schmid E, Winter S, e? al. Vascular smooth musclecells differ from other smooth muscle cells; predominance of vimentin fila- ments and a specific a-type muscle actin. Proc Naf l Acad Sci U S A 1981; 7 8 298-302.

13. Kocher 0, Skalli 0, Cerutti D, Gabbiani F, Gabbiani G. Cytoskeletal features of rat aortic cells during development. An electron micro- scopic, immunohistochemical and biochemical study. Circ Res 1985; 5 6 829-838.

14. Skalli 0, ROprdZ P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Bid 1986; 103: 2781-2796.

15. Hsu SM, Raine L, Farger PH. Use of avidin-biotin peroxidase complex in immunoperoxidase techniques. A comparison between the ABC and unlabelled antibody (PAP) procedure. J Histochem Cytochem 1981; 2 9 557-580.

16. Skalli 0, Gabbiani G, Babai F, Seemayer T, Pizzolato G, Schurch W. Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. 2 . Rhabdomyosarcomas. AmJ Pathol1988; 130 515-531.

17. Dervan PA, Tobbia IN, Casey M, OLoughlin J, OBrien M. Glomus turnours: an immunohistochemical profile of 1 1 cases. Hisropathology 1989: 14483-491.

18. Miettinen M. Antibody specific to muscle actins in the diagnosis and classification of soft tissue tumors. Am J Palhol1988; 130: 205-21 5 .

19. Mittal KR. Gerald W, True LD. Haemangiopericytoma of the breast; report of a case with ultrastructural and immunohistochemical stain- ing.HumPathol1986;17: 1181-1183.

20. Enzinger FM, Weiss SW. Soft Tissue Tumors. 2nd ed. St Louis: C V Mosby, 1988: 475.

21. Skalli 0, Schurch T, Seemayer R, et al. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin iso- forms and intermediate filament proteins. Lab Invesf 1989: 60: 275-285.

22. Schurch W, Skalli 0, Seemayer T, Gabbiani G. Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differen- tiation and origin. I . Smooth muscle tumors. Am J Parhol1987; 128: 91 -103.