Expression of myogenic marker proteins in human leiomyosarcoma

APMIS 105: 793-800. 1997 Copyr igh t 0 A P M I S 1997

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Expression of myogenic marker proteins in human leiomyosarcoma

THOMAS MEYER' and ULRICH BRINCK2

'Center for Internal Medicine and 21nstitute of Pathology, University of Gottingen, Gottingen, Germany

Meyer, T. & Brinck, U. Expression of myogenic marker proteins in human leiomyosarcoma. APMIS 105: 793-800, 1997.

A series of formalin-fixed, paraffin-embedded leiomyosarcomas (n= 1 1) was studied immunohisto- chemically for their expression of various myogenic marker proteins. According to their predominant histological appearance, the tumors were classified as well (n=4), moderately (n= 5 ) , or poorly (n=2) differentiated. Using monoclonal anti-muscle specific actin antibodies from clone HHF35 all examined tumors were positively stained. Desmin was not always found in leiomyosarcomas, since positive staining could be demonstrated only in eight cases. As revealed by staining with anti-vinculin antibodies from clone hVIN-1 using the APAAP technique, all leiomyosarcomas with the exception of one expressed vinculin. Typically, the vinculin imniunoreactivity was detected diffusely throughout the majority of neoplastic cells as well as in vascular smooth muscle cells of blood vessels. Nine leiomyosarcomas displayed a positive staining for calponin, an actin-binding protein expressed in smooth muscle cells and their precursors. The distribution of calponin resembled that of vinculin in decorating myofibrils of nearly all tumor cells. Actinin immunoreactivity was present in tumor cells of all cases, but was expressed also in nontumor cells such as epithelia. These results suggest that the monoclonal antibodies against vinculin and calponin may serve as additional diagnostic markers for myogenic differentiation in leiomyosarcomas and related tumors.

Key words: Immunohistochemistry; leiomyosarcoma; vinculin; calponin.

Thomas Meyer, Center for Internal Medicine, University of Gottingen, Robert-Koch-Str. 40, D-37075 Gottingen, Germany.

Leiomyosarcomas are soft tissue tumors occur- ring preferentially in adult life that by definition exhibit at least some differentiation towards smooth muscle tissue (3). While markers of smooth muscle differentiation are easily docu- mented in some neoplasms, it may be more dif- ficult to find evidence of a myogenic histogenesis in other less well-differentiated tumors. The diagnosis of poorly differentiated, anaplastic leiomyosarcomas presents a challenge for the surgical pathologist, because a broad range of other malignant soft tissue sarcomas share similar histological features (7, 13, 14, 30). Mesenchymal

Received December 6, 1996. Accepted May 20, 1997.

tumors, such as malignant fibrous histiocytoma, fibrosarcoma and malignant schwannoma, and reactive fibroblastic lesions have to be included in the differential diagnosis of smooth muscle neoplasms, since they may all be composed pre- dominantly of fascicles of spindle cells (1, 3, 7, 9, 14, 18, 25, 26, 32).

Histological patterns characterizing well to moderately differentiated leiomyosarcomas comprise cells arranged in bundles with elon- gated or cigar-shaped nuclei surrounded by an eosinophilic cytoplasm (3, 14, 28, 33). Leio- myosarcomas displaying pronounced cellular pleomorphism often lack characteristic histological features, while resembling primitive soft tissue sarcomas. In particular, the histogenesis

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of gastrointestinal stromal tumors remains a persistent source of controversy (5, 7, 11, 19, 21, 26, 29). However, distinguishing leiomyosarcomas from other poorly differentiated tumors is very important with respect to subsequent therapy.

In an attempt to overcome these diagnostic difficulties, immunohistochemical techniques have been introduced in the past few years. Monoclonal antibodies against muscle-specific actin and desmin have proven to be useful tools in the diagnosis of leiomyosarcomas and rhabdomyosarcomas (1, 4, 12, 15-18, 20, 22, 28, 33, 35). Particularly, anti-muscle specific actin antibodies allow the positive identification of tumors derived from a myogenic lineage ( I 8, 35). The diagnostic specificity and sensitivity of anti- desmin antibodies is, however, limited, since some antibodies failed to stain certain subtypes of leiomyosarcomas (1, 3, 10, 17, 22, 25, 27, 34). To positively identify leiomyosarcomas, Lund- gren and co-workers have recommended the use of more than one anti-desmin antibody (15, 16). Immunoreactivity with anti-desmin antibodies has been reported infrequently in some other tumors and nonneoplastic conditions, such as ec- tomesenchymoma, malignant fibrous histiocytoma, rhabdoid and desmoid tumor, Ewing's sarcoma, fibromatosis, and fasciitis (9, 25, 28, 32). Despite fulfilling the morphological criteria of typical leiomyosarcomas, they may sporadi- cally lack both desmin and muscle-specific actin immunoreactivity. However, these tumors must be regarded as malignant smooth muscle neoplasms as other myogenic marker proteins can be detected in them. Recently, it was demonstrated that the monoclonal anti-cortactin antibody 1H1 is a useful reagent in diagnosing leiomyosarcomas (24).

Thus, the aim of our immunohistochemical study was to test monoclonal antibodies to certain cytoskeletal proteins, including vinculin and calponin, for their putative value in the identification of leiomyosarcomas. Since the presence or absence of myogenic marker proteins has been shown to be a useful criterion to distinguish muscle from nonmuscle-derived tumors, it appears attractive to characterize other monoclonal antibodies exhibiting immunoreactivity against muscle-specific proteins respecting their utility as diagnostic aids.

Here we show that the use of antibodies to

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vinculin and calponin provides additional ways of positively identifying leiomyosarcomas, since both reagents strongly recognize their cellular epitopes in the majority of smooth muscle neoplasms even when used in paraffin-embedded material.

MATERIALS AND METHODS

Selection of tissue samples The computerized surgical pathology files at our

University Hospital were reviewed for cases diagnosed as leiomyosarcoma within the last 2 years. The diagnosis was primarily based on standard histo- pathological criteria requiring features of a malignant smooth muscle tumor with increased mitotic activity, hypercellularity, nuclear pleomorphism and atypia. Histological slides from 1 1 cases in which formalin-fixed paraffin blocks were available and in which adequate tissue remained in the block were re- examined. Histological specimens were studied by light microscopy for malignancy grade, number of mitotic figures per 10 high-power fields, and presence of necrosis.

Irnmunohistochernical stainings From all paraffin-embedded materials. sections 3

pm in diameter were recut and mounted on slides co- ated with poly-L-lysine (Sigma, Deisenhofen. Ger- many). Sections were deparaffinized in xylene. rehy- drated in graded alcohol, and immersed in deionized water. After rinsing in Tris-buffered saline (TBS: 150 mM NaCI, 50 mM Tris-HC1, pH 7.6) sections were incubated with primary antibodies in moist chambers for 45 min at room temperature. All antibodies were diluted in RPMI 1640 (Gibco Laboratories. Grand Island, NY, USA) supplemented with lo'%, fetal calf serum (FCS). Dilutions and sources of monoclonal antibodies were as follows: anti-desmin clone DE-R- 11 (Dakopatts, Glostrup, Denmark) diluted 1:100, anti-a-sarcomeric actin clone HHF35 (Dakopatts) used at a concentration of 1: 100, anti-vinculin clone hVIV-1 (Sigma) diluted 1 :400, anti-actinin clone BM- 752 (Sigma) diluted 1:200, anti-calponin clone hCP (Sigma) used at a concentration of 1:1000, and anti- Ki-67 clone MIB I (Dakopatts). Negative controls were carried out by substituting nonimmune mouse serum for primary antibody solutions. In two cases not stained by conventional techniques, antigen retrieval was performed by microwaving the sections for 2 x 4 min in a commercially available buffer (Dakopat t s).

After reacting with primary antibodies, sections were washed three times in TBS followed by incuba- tion with rabbit anti-mouse immunoglobulins (Dako- patts) diluted 1:25 in RPMI+ 10% FCS for 30 min at room temperature. Sections were then rinsed in TBS

MARKER PROTEINS IN LEIOMYOSARCOMA

and incubated with alkaline phosphatase-conjugated mouse monoclonal anti-alkaline phosphatase (Dako- patts) diluted 1:25 in RPMI+lO% FCS.

Alkaline phosphatase activity was visualized using the New Fuchsin method. Briefly, a freshly made solution of 50 mM 2-amino-2-methyl- 1,3-propanedi- 01 (Merck, Darmstadt, Germany), 1.7 mM levamiso- le (Sigma), 150 mM NaC1, 40 mM Tris, pH 9.7, was mixed with vol naphthol-AS-BI phosphate (Flu- ka. Neu-Ulm, Germany) dissolved in N,N-dimethyl- formamide. A freshly prepared solution containing 5%) New Fuchsin (Sigma) and 3% NaN02 was added

vol), the pH adjusted to 8.7 by adding HCl, and the final solution mixed through a filter paper directly on the slides. After stopping the reaction with dis- tilled water, samples were counterstained for 5 min in Mayer's hematoxylin, and coverslips were applied in a synthetic mounting medium.

Gruding Tumor cells were considered positive if a discrete

red cytoplasmic staining greater than background and distant from the edge of the section or areas of necrosis could be visualized. The extent of immuno- staining in each specimen was semiquantitatively graded on the following scale: - no staining; + slight or <10'%1 tumor cell staining; + + moderate or 10% to 50% tumor cell staining; + + + intensive or >50% tumor cell staining.

RESULTS

Formalin-fixed, paraffin-embedded tissue from 11 patients diagnosed as having leiomyosarcoma was included in our retrospective archival study. The mean age of the patients at the time of diagnosis was 72 years, range 61 to 87 years (Table 1). There were seven females and four males (F-M ratio 1.75:l). The anatomic location of the tumor masses included the mesentery (n=3, with one metastasis from the left

ovary), skin (n=3), small intestine bowel (n=2), retroperitoneum (n=2), and endometrium (n= 1). Light microscopy performed on hematoxylin-eosin stained sections showed that tissue from all leiomyosarcomas consisted of spindle- shaped cells with abundant eosinophilic cytoplasm (Fig. 1). Typically, tumor cells were vari- ably arranged in interweaving fascicles. Ne- crosis was present in six cases. Seven of the examined cases had a mild to moderate degree of cellular pleomorphism. Cross-striations typically present in rhabdomyosarcoma were never observed. According to the predominant histological appearance in hematoxylin-eosin stains, leiomyosarcomas were classified as well (n = 4), moderately (n=5), or poorly (n=2) differentiated. Mitotic activity was generally frequent in all tumors, reaching up to four division figures per high-power field. There were more mitotic figures in poorly differentiated than in differentiated neoplasms. Tumors studied immuno- histochemically for the expression of the pro- liferation marker MIB I showed a significantly increased rate of proliferative activity, ranging up to 3040% positively stained nuclei (Fig. 2).

When tested for the expression of muscle-specific actin, tumor cells in all specimens showed a high proportion of positively stained cells (Fig. 3). In contrast, the immunoreactivity of desmin was confined to a smaller percentage of tumor cells (Fig. 4). Only in 8 of 11 neoplasms could the intermediate filament protein desmin be detected, while it was totally lacking in 3 leiomyosarcomas. In our series, the percentage of cells positive for desmin was more variable compared to the uniform distribution of muscle-specific actin. Whereas in most cases muscle-specific actin presented diffusely

TABLE 1 . Expression of myogenic proteins in 11 cases of leiomyosurcoma Case Age Sex Tumor location Grade Vinculin Calponin Actinin Actin Desmin

1 77 F Mesentery 1 + - + ++ + 2 57 M Retroperitoneum 3 ++ ++ +++ ++ 3 69 F Skin (dermal) 1 +++ ++ +++ ++ 4 69 F Retroperitoneum 1 + ++ +++ ++ + 5 87 M Skin (dermal) 2 + + +++ ++ 6 71 F Intestine 2 - ++ ++ ++ + 7 61 F Mesentery 2 +++ +++ +++ ++ ++ 8 63 M Intestine 2 + ++ +++ ++ ++ 9 80 F Mesentery 2 + +++ ++ ++ +

-

-

-

10 81 F Uterus 3 + - ++ ++ + 1 1 78 M Skin (subcutaneous) 1 + ++ + ++ +

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Fig. 1. Microphotograph of a leiomyosarcoma composed of fascicles of smooth muscle cells demonstrat- ing mitotic activity (Hematoxylin-eosin stain). Fig. 2. Demonstration of proliferative activity in a leiomyosarcoma by staining with the monoclonal anti-Ki-67 antibody MIB I. Note the increased expression of this nuclear protein in proliferating tumor cells.

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throughout the tumor tissue, desmin was demonstrated more focally in the majority of leiomyosarcomas. The presence or absence of desmin staining was not related to the histological grade of the tumor, as all three leiomyosarcomas lacking desmin expression ranged from grade 1 to 3. Reactivity for desmin appeared to be most obvious in densely cellular areas.

By using the monoclonal anti-vinculin antibody hVIN-1, all malignant smooth muscle tumors except one were shown to have a positive staining pattern. The vinculin immunoreactivity was typically scattered throughout the majority of tumor cells (Fig. 5 ) . Since smooth muscle cells of blood vessels were intensely decorated by anti-vinculin antibodies, the detection of positive vascular labeling provided a good and reliable internal standard for an optimal staining procedure. In two cases, antigen retrieval techniques had to be used, as both had tumor vessels which exhibited only a weak positive staining, unless the sections were first processed for microwaving. The only tumor (case 6) negative for vinculin was a moderately differentiated leiomyosarcoma located in the small intestine. Since this tumor exhibited typical histological features of a leiomyosarcoma and labeled positively for muscle-specific actin and desmin, there could be no doubt that it orginally derived from neoplastic smooth muscle tissue.

The monoclonal anti-calponin antibody hCP yielded strong immunolabeling in 9 of 11 leiomyosarcomas. As expected for an actin-binding protein, reactivity was exclusively confined to the cytoplasm (Fig. 6). All neoplasms exhibiting positivity for calponin showed a diffuse homo- geneous staining with this antibody. The two tumors lacking detectable calponin expression were a mesenteric metastasis of an ovarian leio-

myosarcoma and an endometrial stromal sarcoma, both of which displayed muscle-specific actin and desmin staining.

Regardless of the histological grade, actinin was detected in all examined leiomyosarcomas (Fig. 7). However, expression of actinin was not restricted to tumor cells, as shown by its presence in non-neoplastic cells such as epithelia.

DISCUSSION

As histological patterns obtained in conventional stainings do not necessarily represent valuable indicators of cellular differentiation in certain mesenchymal tumors, in order to reach the correct diagnosis it is important to extend the morphological evaluation by means of a detailed immunohistochemical study. The cur- rently used classification scheme of soft tissue sarcomas is dependent on the histological detection of certain marker proteins, thereby demon- strating the histogenic origin of a tumor. Since histogenetically different neoplasms sometimes have similiar if not identical morphological features, the differential diagnosis of malignant sarcomas has to take into account the tissue- specific expression of certain differentiation- linked proteins. It is generally accepted that tumors with a common histogenesis often exhibit a broad spectrum of histological features. In particular, leiomyosarcomas are known to dis- play a wide range of variable morphological patterns even within the same tumor mass, rein- forcing the hypothesis of a histogenetic hetero- geneity for this tumor entity (15, 16, 21, 30). The varying immunohistochemical properties of leiomyosarcomas seem to indicate a variable expression of myogenic marker proteins depend-

Fig. 3. Detection of muscle-specific actin in a leiomyosarcoma as revealed by immunohistochemical staining with the monoclonal antibody HHF35 using APAAP technique. Fig. 4. Microphotograph of a leiomyosarcoma displaying several tumor cells that stain positively for desmin (APAAP using anti-desmin antibody clone DE-R-11). Fig. 5. Immunohistochemical detection of vinculin in a leiomyosarcoma as revealed by staining with monoclonal antibodies from clone hVIN- 1 (APAAP). Note that vinculin is expressed diffusely throughout the tumor tissue. Fig. 6. Leiomyosarcoma showing calponin staining as revealed by monoclonal anti-calponin antibody hCP (APAAP). Calponin is abundantly expressed in tumor cells.

Fig. 7. Typical pattern of actinin expression in a leiomyosarcoma as obtained by immunohistochemical staining using anti-actinin antibody from clone BM-752.

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ing on the anatomic sites and histogenetic origin.

It is surprising that a detailed investigation of the expression pattern of myogenic marker proteins, including vinculin and calponin in leiomyosarcomas, has not so far been under- taken. In contrast, smooth muscle differentiation has been analyzed in leiomyomas, the most common benign variants of smooth muscle neoplasms (6, 27). It has been reported in uterine leiomyomas that the expression of caldesmon, vinculin, desmin, a-smooth muscle actin and calponin resembled that of normal myometrium with the exception of a low- molecular-weight isoform of calponin which was selectively expressed in normal uterine tissue (2).

However, comparable data for the expression profile of these cytoskeletal proteins in malignant tumor variants of the leiomyogenic lineage is not available. Thus, we focused our attention on the phenotypical characterization of leiomyosarcomas by using a panel of monoclonal antibodies against myogenic marker proteins.

Unfortunately, antibodies against caldesmon failed to react with paraffin-embedded tissue and therefore had to be excluded from our study. Monoclonal antibodies against the myogenic intermediate filament protein desmin have been widely used as diagnostic tools for the positive identification of rhabdomyosarcomas and leiomyosarcomas as they stain sarcomeric cells and their precursors as well as visceral and some vascular derived smooth muscle cells (12, 17, 20, 25, 28, 33). Our results concerning desmin expression are in line with other data showing that within a given leiomyosarcoma the numbers of desmin-positive cells are often far fewer than those expressing muscle-specific actin ( 5 , 10, 28, 35). In our series, desmin immunoreactivity was found in 8 of 11 tumors, confirming previous observations that lack of desmin expression does not exclude the diagnosis of leiomyosarcoma (18, 28, 35).

Here we demonstrate that for the detection of leiomyosarcomas monoclonal anti-vinculin and anti-calponin antibodies exhibited an increased sensitivity relative to antibodies against desmin. With one exception, all examined smooth muscle tumors revealed the presence of immunodetectable vinculin. While desmin antibodies typically reacted only with a subset of

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neoplastic smooth muscle cells, vinculin immunoreactivity was found diffusely distributed throughout the tumor tissue.

Vinculin is a cytoskeletal protein associated with membrane actin-filament-attachment sites of cell-cell and cell-matrix adherent-type junc- tions (8, 23). In normal tissue, vinculin is localized in dense plaques of smooth muscle, in in- tercalated disks and costameres of cardiac muscle, and in focal contacts of cultivated cells (8, 23). It is also present in cells that are not organized as solid tissues, such as platelets (23). During cultivation, smooth muscle cells con- tinue to express the low-molecular-weight isoform of vinculin (1 30 kDa), while losing meta- vinculin (1 50 kDa) and the intermediate filament protein desmin, which is replaced by vi- mentin (31).

Calponin is a 34 kDa actin-binding protein known to inhibit the actomyosin ATPase, sug- gesting a regulatory function in contraction (6). In normal tissue, calponin expression is restricted to smooth muscle, where it can be found in large amounts (6). As shown in our study, a positive staining for calponin was achieved in 9 of 11 cases of leiomyosarcoma. Calponin was localized diffusely in the majority of all tumor cells, although displaying varying degrees of expression between individual tumors.

Vinculin expression could not be demonstrated in Wilms’ tumors, malignant schwannomas, neuroblastomas or extraosseous Ewing’s sarcomas. In benign tumors of soft tissue, such as subcutaneous fibromas, neuro- fibromas, and palmar fibromatosis, we found no vinculin staining, even when antigen retrieval techniques were used. Since calponin, like vinculin, was also detected in rhabdomyosarcomas (two of four rhabdomyosarcomas expressed calponin), it can not be regarded as a specific marker protein identifying exclusively smooth muscle-derived tumors (data not shown). How- ever, the immunohistochemical detection of vinculin and calponin in the majority of leiomyosarcomas suggests that both cytoskeletal proteins may serve as diagnostic markers for myogenic differentiation. An extensive study of the reactivity of anti-vinculin and anti-calponin antibodies on paraffin-embedded tumor material other than of muscle origin has not yet been performed. Our results suggest, however,


that vinculin and calponin may be useful ad- juncts to other myogenic proteins in the study and diagnosis of leiomyosarcoma and related lesions.

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Expression of myogenic marker proteins in human leiomyosarcoma

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