H I S T O CH E MI CA L CH A RA C TER IS TIC S O F M UC OSUBSTANCES I N T H R E E S OF T-TIS S UE T U M O U R S M. I. FILIPE AND D. H. MACKENZIE Department of Histopathology, Westminster Medical School, London
H I S T o c H EM I c A L studies may provide useful diagnostic information in the study of soft-tissue tumours (Tighe and Meachin, 1962; Tighe, 1963; Enzinger, 1965; Fine and Horn, 1969). One of the properties shared by a number of such tumours is the ability to produce varying amounts of mucosubstances which give the so-called myxoid appearance noted both macroscopically and microscopically. Three entities in which large amounts of mucosubstances may be found are the myxoid liposarcoma (Enzinger and Winslow, 1962); some malignant fibrous histiocytomas (Kempson and Kyriakos, 1972); and the intramuscular myxoma (Enzinger, 1965). In the present study an attempt was made to identify the mucosubstances produced and to note similarities and differences in the three conditions. MATERIALS AND METHODS Twenty-seven cases were studied consisting of myxoid liposarcoma (I 6 cases), malignant fibrous histiocytoma (5 cases) and intramuscular myxoma (6 cases). In all cases haematoxylin and eosin sections indicated the presence of extensive myxoid areas. The material had been fixed in 10 per cent. formol saline and embedded in paraffin. Serial sections cut at 5 p m were stained with PAS with and without diastase, alcian blue at pH 2.5 andpH 1.0 and in azur A at pH 2.0 and p H 4.0 as described by Pearse (1968). They were also stained with alcian blue at p H 5.7 plus MgClz according to the method of Scott and Dsrling (1965) and by high irondiamine (Spicer, 1965). Parallel sections were pre-incubated with testicular hyaluronidase while controls were pre-incubated in buffer solution alone. The results are shown in the table.
DISCUSSION A study of the table indicates that some differences exist between the mucosubstances found in myxoid liposarcomas and those found in the other two conditions. Myxoid liposarcomas contained neutral mucosubstances which were absent from the malignant fibrous histiocytomas and intramuscular myxomas. All three types of tumour contained acid mucosubstances, but sulphated radicals were less common in myxoid liposarcomas. Testicular hyaluronidase digestion abolishes the staining reactions with alcian blue at pH 2.5 and 1.0, with azur A at pH 4 and with high iron-diamine. However, this enzyme removes both hyaluronic acid and some sulphated mucosubstances such as chondroitin sulphate A and C from the tissues and does not distinguish between these substances. We appreciate that the results must be interpreted with caution because the tissues had been preserved in 10 per cent. formalin Received 5 Feb. 1975; accepted 24 Feb. 1915. J. ?A781.-VOL.
118 (1976)
17
B
(12.5 %I
Some sulphated MS. Positive (metachromasia) Carboxyl groups of hyaluronic 68 % acid. Sialomucins
Azur A pH 4 0
Positive (brown-black)
Negative
Strongly sulphated MS
Azur A pH 2.0
Most sulphated MS
Not done
Sulphated MS with MgCl2 concentration above 0 . 2 ~
Alcian blue p H 5.7+ MgClz
High iron-diamine
Positive (basophilia) 37%
Strongly sulphated MS
Alcian blue pH 1.0
Weakly sulphated MS. Positive (basophilia) 100% Carboxyl groups of hyaluronic acid. Sialomucins
Alcian blue pH 2.5
Positive (red) 75 %
Myxoid liposarcoma
Neutral MS
Mucosubstances (MS) visualised
PAS fdiastase
Method
Negative
Intramuscular myxoma
(40%)
Positive (brown-black
60%
Positive (metachromasia)
Negative
Positive (basophilia) 80%
Positive (basophilia) 80 %
Positive (brown-black) (60%I
F;
8
Kj
0
5
3 Positive (basophilia) 83 % Negative Positive (metachromasia) 66 %
b
2
tr
%
Positive (basophilia) 50%
Positive (basophilia) 100% Positive (basophilia) 100%
Negative
Malignant fibrous histiocytoma
Histochemical characteristics of mucosubstances in three soft-tissue tumurs
TABLE
MUCOSUBSTANCES IN TUMOURS
19
for varying lengths of time. Various fixatives have been used for the preservation of carbohydrate substances in connective tissue (Spicer et al., 1965). Fixation in 10 per cent. buffered formol-calcium for 24 hr at 4°C is satisfactory. Another way of preventing diffusion of tissue components without the disadvantages of blocking the histochemical reactions of the mucosubstances by the fixative, is the use of fresh freeze-dried material fixed in paraformaldehyde vapour. It is hoped to carry out further investigations using these methods. However, in spite of the fixation difficulty in the present series, it seems unlikely that histochemical studies will contribute significantly to the differentiation of the three tumour entities considered here. SUMMARY
The mucosubstances present in myxoid liposarcomas, malignant fibrous histiocytomas and intramuscular myxomas were investigated. The results suggest that such investigations are unlikely to provide a useful diagnostic aid in these three lesions. We are indebted to our clinical colleagues at Westminster Hospital for permission to study their cases. Addendum
Since the manuscript was submitted Kindbloom, L. and Angervall, L. have performed similar studies (Cancer, 1975, 36, 985). REFERENCES ENZINGER, F. M. 1965. In Tumours of bone and soft tissues, 8th Annual Clinical Conference on Cancer 1963, University of Texas, p. 321, Chicago Year Book Medical Publishers. F. M. 1965. Intramuscular myxoma. Amer. J . Clin. Path., 43, 104. ENZINGER, ENZINGER, F. M., AND WINSLOW, D. 1962. Liposarcoma. Virchows Arch. Path. Anat., 335, 367. FINE, G., AND HORN, R. C. 1969. Soft tissue tumours: aids in differential diagnosis. Pathology annual. Appleton Century-Crofts, New York, U.S.A., p. 211. KEMPSON, R., AND KYRIAKOS, M. 1972. Fibroxanthosarcoma of the soft tissues. Cancer, 29, 961. RARSE, A. G . E. 1968. In Histochemistry, theoretical and applied, 3rd ed., J. A. Churchill Ltd, London, vol. 1. TIGHE, J. R. 1963. The histological demonstration of mucopolysaccharides in connective tissue tumours. J. Path. Bact., 86,41. TIGHE, J. R., AND MEACHIN,G. 1962. Sulphate utilisation by mucinous and other connective tissue tumours. J. Path. Bact., 83, 195. Scorn, J. E., AND DORLING,J. 1965. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie, 5 , 221. SPICER,S. S. 1965. Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem., 13, 211. SPICER,S . S., HORN,R. G., AND LEPPI,T. J. 1967. In Symposium on connective tissue research methods. International Academy of Pathology Monograph. Williams and Wilkins Co., Baltimore, U.S.A., p. 251.
H I S T o c H EM I c A L studies may provide useful diagnostic information in the study of soft-tissue tumours (Tighe and Meachin, 1962; Tighe, 1963; Enzinger, 1965; Fine and Horn, 1969). One of the properties shared by a number of such tumours is the ability to produce varying amounts of mucosubstances which give the so-called myxoid appearance noted both macroscopically and microscopically. Three entities in which large amounts of mucosubstances may be found are the myxoid liposarcoma (Enzinger and Winslow, 1962); some malignant fibrous histiocytomas (Kempson and Kyriakos, 1972); and the intramuscular myxoma (Enzinger, 1965). In the present study an attempt was made to identify the mucosubstances produced and to note similarities and differences in the three conditions. MATERIALS AND METHODS Twenty-seven cases were studied consisting of myxoid liposarcoma (I 6 cases), malignant fibrous histiocytoma (5 cases) and intramuscular myxoma (6 cases). In all cases haematoxylin and eosin sections indicated the presence of extensive myxoid areas. The material had been fixed in 10 per cent. formol saline and embedded in paraffin. Serial sections cut at 5 p m were stained with PAS with and without diastase, alcian blue at pH 2.5 andpH 1.0 and in azur A at pH 2.0 and p H 4.0 as described by Pearse (1968). They were also stained with alcian blue at p H 5.7 plus MgClz according to the method of Scott and Dsrling (1965) and by high irondiamine (Spicer, 1965). Parallel sections were pre-incubated with testicular hyaluronidase while controls were pre-incubated in buffer solution alone. The results are shown in the table.
DISCUSSION A study of the table indicates that some differences exist between the mucosubstances found in myxoid liposarcomas and those found in the other two conditions. Myxoid liposarcomas contained neutral mucosubstances which were absent from the malignant fibrous histiocytomas and intramuscular myxomas. All three types of tumour contained acid mucosubstances, but sulphated radicals were less common in myxoid liposarcomas. Testicular hyaluronidase digestion abolishes the staining reactions with alcian blue at pH 2.5 and 1.0, with azur A at pH 4 and with high iron-diamine. However, this enzyme removes both hyaluronic acid and some sulphated mucosubstances such as chondroitin sulphate A and C from the tissues and does not distinguish between these substances. We appreciate that the results must be interpreted with caution because the tissues had been preserved in 10 per cent. formalin Received 5 Feb. 1975; accepted 24 Feb. 1915. J. ?A781.-VOL.
118 (1976)
17
B
(12.5 %I
Some sulphated MS. Positive (metachromasia) Carboxyl groups of hyaluronic 68 % acid. Sialomucins
Azur A pH 4 0
Positive (brown-black)
Negative
Strongly sulphated MS
Azur A pH 2.0
Most sulphated MS
Not done
Sulphated MS with MgCl2 concentration above 0 . 2 ~
Alcian blue p H 5.7+ MgClz
High iron-diamine
Positive (basophilia) 37%
Strongly sulphated MS
Alcian blue pH 1.0
Weakly sulphated MS. Positive (basophilia) 100% Carboxyl groups of hyaluronic acid. Sialomucins
Alcian blue pH 2.5
Positive (red) 75 %
Myxoid liposarcoma
Neutral MS
Mucosubstances (MS) visualised
PAS fdiastase
Method
Negative
Intramuscular myxoma
(40%)
Positive (brown-black
60%
Positive (metachromasia)
Negative
Positive (basophilia) 80%
Positive (basophilia) 80 %
Positive (brown-black) (60%I
F;
8
Kj
0
5
3 Positive (basophilia) 83 % Negative Positive (metachromasia) 66 %
b
2
tr
%
Positive (basophilia) 50%
Positive (basophilia) 100% Positive (basophilia) 100%
Negative
Malignant fibrous histiocytoma
Histochemical characteristics of mucosubstances in three soft-tissue tumurs
TABLE
MUCOSUBSTANCES IN TUMOURS
19
for varying lengths of time. Various fixatives have been used for the preservation of carbohydrate substances in connective tissue (Spicer et al., 1965). Fixation in 10 per cent. buffered formol-calcium for 24 hr at 4°C is satisfactory. Another way of preventing diffusion of tissue components without the disadvantages of blocking the histochemical reactions of the mucosubstances by the fixative, is the use of fresh freeze-dried material fixed in paraformaldehyde vapour. It is hoped to carry out further investigations using these methods. However, in spite of the fixation difficulty in the present series, it seems unlikely that histochemical studies will contribute significantly to the differentiation of the three tumour entities considered here. SUMMARY
The mucosubstances present in myxoid liposarcomas, malignant fibrous histiocytomas and intramuscular myxomas were investigated. The results suggest that such investigations are unlikely to provide a useful diagnostic aid in these three lesions. We are indebted to our clinical colleagues at Westminster Hospital for permission to study their cases. Addendum
Since the manuscript was submitted Kindbloom, L. and Angervall, L. have performed similar studies (Cancer, 1975, 36, 985). REFERENCES ENZINGER, F. M. 1965. In Tumours of bone and soft tissues, 8th Annual Clinical Conference on Cancer 1963, University of Texas, p. 321, Chicago Year Book Medical Publishers. F. M. 1965. Intramuscular myxoma. Amer. J . Clin. Path., 43, 104. ENZINGER, ENZINGER, F. M., AND WINSLOW, D. 1962. Liposarcoma. Virchows Arch. Path. Anat., 335, 367. FINE, G., AND HORN, R. C. 1969. Soft tissue tumours: aids in differential diagnosis. Pathology annual. Appleton Century-Crofts, New York, U.S.A., p. 211. KEMPSON, R., AND KYRIAKOS, M. 1972. Fibroxanthosarcoma of the soft tissues. Cancer, 29, 961. RARSE, A. G . E. 1968. In Histochemistry, theoretical and applied, 3rd ed., J. A. Churchill Ltd, London, vol. 1. TIGHE, J. R. 1963. The histological demonstration of mucopolysaccharides in connective tissue tumours. J. Path. Bact., 86,41. TIGHE, J. R., AND MEACHIN,G. 1962. Sulphate utilisation by mucinous and other connective tissue tumours. J. Path. Bact., 83, 195. Scorn, J. E., AND DORLING,J. 1965. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie, 5 , 221. SPICER,S. S. 1965. Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem., 13, 211. SPICER,S . S., HORN,R. G., AND LEPPI,T. J. 1967. In Symposium on connective tissue research methods. International Academy of Pathology Monograph. Williams and Wilkins Co., Baltimore, U.S.A., p. 251.
