Infantile Digital Fibromatosis

Ultrastructural, Histochemical, and Tissue Culture Observations

HlROSHl IWASAKI, MD,* MASAHIRO KIKUCHI, MD,t RYOlCHl MORI, MD,* JUNK0 MIYAZONO, BS,§ MUNETOMO ENJOJI, MD,Il NOR10 SHINOHARA, MD,ll AND AKlO MATSUZAKI, MD#

Three cases of infantile digital fihromatosis were studied by electron microscopy, enzyme histochem- istry, and tissue culture. The tumors were made up equally of myofibroblasts containing electron-dense inclusions which were composed chiefly of microfilaments measuring about 5 to 7 nm. Dense bodies usually observable in the smooth muscle cells were found in the bundles of these microfilaments and in the processes of the inclusions, suggesting that these inclusions may represent an abnormal ac- cumulation of contractile protein in the cytoplasm of tumor cells. Two cell lines were established from culture of the tumor cells, and the cultured cells also contained inclusion bodies showing the same morphologic characteristics as those of the original tumor cells. Lysosomal enzymes were abundant in the cultured cells, but they were scant in the cells of the fresh tissue specimens. Cocultivation of the cultured cells with human embryonic lung cells yielded no cytopathic effect.

Cancer 46:2238-2247, 1980.

NFAN I I L E DIGITAL FIBROMATOSIS is a peculiar I fibrous growth that may be singular or multiple on the fingers and toes of infants and children. The growth may recur, but it never metastasizes. It is also char- acterized by the presence of eosinophilic cytoplasmic inclusion bodies. 1 , 3 , 1 2 , 1 7 3 1 9

Ultrastructural studies have confirmed the presence of inclusion bodies in the cytoplasm of tumor

clusion, has been a subject of controversy. Although some authors have suggested a viral i n fec t i~n ,* , '~ , ' ~ no

cells ,2,4,3,6,X,9,I1-15.*",~* but the true nature of the in-

From the Department of Pathology and the Department of Orthopedics, Fukuoka University School of Medicine: the Depart- ment of Virology and the Department of Pathology, Faculty of Medicine, Kyushu University: and the Department of Orthopedics, National Fukuoka Central Hospital, Fukuoka, Japan.

* Associate Professor of Pathology, Fukuoka University School of Medicine.

t Professor ofPathology, Fukuoka University School of Medicine. $ Professor of Virology, Faculty of Medicine, Kyushu University. 9 Fellow in Virology, Faculty of Medicine, Kyushu University. l 1 Professor of Pathology, Faculty of Medicine, Kyushu University. '1 Head of Orthopedics, National Fukuoka Central Hospital. # Associate Professor of Orthopedics, Fukuoka University

School of Medicine. Address for reprints: Hiroshi Iwasaki, MD, Department of

Pathology, Fukuoka University School of Medicine, 34 Nanakuma, Nishiku, Fukuoka 814, Japan.

The authors thank Dr. M. Miyoshi, MD, Professor of Anatomy, Fukuoka University School of Medicine, and Dr. H. Ishikawa, MD, Associate Professor of Anatomy, Faculty of Medicine, Uni- versity of Tokyo, for their valuable advice in electron microscopic studies: and Dr. T. Imai, MD, Professor of Pathology, Fukuoka University Hospital, for his valuable advice and encouragement.

Accepted for publication December 5 , 1979.

conclusive evidence has so far been obtained. In 1979 Bhawan et a/.* pointed out that the lesion consisted of typical myofibroblasts which contained inclusion bodies probably derived from degradation products of actomyosin. The present article describes three new cases of infantile digital fibromatosis studied by elec- tron microscopy, tissue culture, and histochemistry.

Materials and Methods

During the period from April 1977 through March 1979, three cases of infantile digital fibromatosis were examined at the Department of Pathology, Fukuoka University School of Medicine. Clinical data of these cases are summarized in Table 1.

Light Microscopy

Paraffin sections of tumor tissue were stained with hematoxylin and eosin (H & E), Masson's trichrome, elestica van Gieson, alcian blue, periodic acid-Schiff (PAS) with or without diastase digestion, phos- photungstic acid-hematoxylin, Lendrum's phloxine- tartrazine, methyl green-pyronin, and Feulgen's method. The same stains were applied to the cells cultured on coverglass.

Electron M ic Y O s c op y

Portions of fresh surgical specimens were im- mediately fixed in 3% glutaraldehyde in 0.1 M

0008-S43)3/80/ I 1 1512238 $1 .OO 0 American Cancer Society

2238

No. 10 INFANTILE DIGITAL FIBROMATOSIS . Zwasaki et al . 2239

cacodylate buffer at pH 7.4, and then postfixed in 1% buffered osmium tetroxide. Following dehydration in a graded series of alcohol, blocks were embedded in epoxy resin (Epon 812). The cell pellets obtained from tissue culture were processed in a similar way. Areas containing inclusions were selected from 1-pm sections stained with 1% toluidine blue, and ultrathin sections stained with uranyl acetate and lead citrate were examined on an electron microscope.

Tissue Culture and Virological Study

Parts of excised tumors were minced, treated with 0.25% trypsin at 37 C for 60 minutes, suspended in Eagle’s MEM supplemented with 10% calf serum, and cultured in plastic dishes (Falcon #3001) kept in an incubator with humidified 5% CO, atmosphere. After the second passage, subcultures were made in bottles every 4-5 days at a splitting ratio of 1.2. For viro- logic study, the cells obtained from the subculture were cocultivated with human embryonic lung cells to ob- serve the cytopathic changes.

Enzyme Histochrmistry

In each case enzyme histochemical studies were carried out using frozen sections of fresh tumor tissue and the cells obtained from tissue culture. The en- zymes thus investigated included naphthol-ASD- acetate esterase, a-naphthyl acetate esterase, adenosine triphosphatase, acid phosphatase, 5’-nucleotidase, P-glucuronidase, and alkaline phosphatase.

Results

Light Microscopy

Each tumor consisted of moderately cellular, inter- lacing bundles of fibroblast-like tumor cells embedded in an abundant collagenous matrix. The recurrent tumors showed an increased amount of collagen com- pared with the initial tumors. Each tumor cell had an elongated nucleus with one or two distinct nucleoli, and variable amounts of amphophilic cytoplasm with indistinct outline.

Eosinophilic inclusions were found within the cytoplasm of many tumor cells, often adjacent to one pole of the nucleus (Fig. 1). The inclusion bodies could be easily recognized in the sections stained with H & E: they were round or oval, pinkish bodies measuring about 1.5 to 10 pm in diameter, with an average of 3.6 pm. They stained bright red with Masson’s trichrome, yellow with van Gieson, purple with PTAH, and bright red with Lendrum’s phloxine- tartrazine. The inclusion bodies were negative for alcian blue, PAS, methyl green-pyronin, or Feulgen’s

TABLE 1. Clinical Data for Three Patients with Infantile Digital Fibromatosis

Age Dura- Follow-~p NO. of Case at tion Site and time recur- no. Sex onset (mo.) size (mm) (mo.) rences

1 F Birth 7 Dorsum distal 10 0 phalanx

R. 2nd toe 11 x 10 x 7

2 M 1 mo. 3 Dorsum distal 30 2 phalanx

R. 2nd toe 6 ~ 6 x 4

3 F 8 mo. 4 Dorsum middle 8 1 phalanx

L. 3rd finger 15 x 12 x 5

Electron Microscopy

All tumors from the three patients were essentially identical in ultrastructural features. They were com- posed of elongated cells separated by abundant collagen fibrils and amorphous ground substance (Fig. 2). Most tumor cells had ultrastructural features of myofibroblasts. The cytoplasm contained filamentous structures of two different types: (1) microfilaments measuring 5-7 nm in diameter: and (2) filaments measuring about 10 nm in diameter. In addition,

FIG. 1. Case 1. The tumor consists of fibroblast-like cells embedded in an abundant collagenous matrix. Inclusion bodies are noted in the cytoplasm of tumor cells. often adjacent to one pole of the

reagent. nuclei (H E, ~ 5 5 0 ) .

2240 CANCER November 15 1980 Vol. 46

FIG. 2. Case 1. A tumor cell in the upper portion has an intracytoplasmic inclusion. Part of the cell surface is covered by basement membrane. An extravasated red cell is found adjacent to the tumor cell. The cells in the middle and lower portions contain dense bodies such as seen in smooth muscle cells. Note well-developed granular endoplasmic reticulum and Golgi apparatus ( x 5,900).

No. 10 INFANTILE DIGITAL FIBROMATOSIS . Ibtusaki et ul. 224 1

FIG. 3. Case 1. A tumor cell with the feature of myofibroblast has an inclusion extending a strap-like process containing many dense bodies. Note indented nucleus, rich network of granular endoplasmic reticulum, and well-developed Golgi zone (~8,800) .

2242 CANCER November 15 1980 Vol. 46

FIG. 4. (c1hoi.e): Detail of the inclusion with strap-like process in Fig. 3. Dense bodies are scattered in the process consisting of microfilaments ( x 16,600).

FIG. 5. ( h c l o ~ ) : Case 2. Part of an inclusion body containing membrane-bound vesicles. A thick bundle of microfilaments adjacent to the inclusion contains several dense bodies (~20,000).

No. 10 INFANTILE DIGITAL FlBROMATOSlS Zwrsaki et al . 2243

FIG. 6 . Case 1. Inclusion and its process arc composed chiefly of microfilaments measuring 5-7 nm in diameter. In addition, filaments of 10 nm in diameter are noted at the periphery of the inclusion and in the surrounding cytoplasm (X42,700).

microtubules measuring about 24 nm in diameter were found in the cytoplasm. The above-mentioned micro- filaments were arranged in bundles running parallel to the long axis of tumor cells and were often attached to the cell membrane. Dense bodies usually seen in the ordinary smooth muscle cells were found to be inter- spersed along the bundles of the microfilaments (Figs. 3, 4, 5). The 10 nm filaments were not arranged in bundles, but were randomly distributed in the cytoplasm (Fig. 6).

The most characteristic feature was the presence in the cytoplasm of tumor cells of electron dense, non- membrane-bound, spherical inclusions. These in- clusions were composed chiefly of closely packed microfilaments which measured 5-7 nm and had a resemblance to those which were arranged in bundles interspersed with dense bodies. The inclusions were not uniform in size, ranging from 1,000 to 10,000 nm in diameter. The smaller inclusions had an irregular poorly defined border, while the larger inclusions ap- peared round and were sharply demarcated. At the periphery of these inclusions were seen prominent felt-like filamentous structures. More centrally, how- ever, the filaments were so closely packed that they

gave an amorphous granular appearance. The 10 nm filaments were seen most abundantly in the cytoplasm surrounding the inclusions, but sometimes they were also found within the inclusions themselves, mainly at their periphery (Fig. 6). The larger inclusions often contained membrane-bound small vesicles, probably derived from the entrapped cell organelles (Fig. 5) . Strap- or finger-like processes were frequently found to be extending from the inclusions. These processes showed an essentially same structure as those of the bundles of microfilaments and sometimes contained dense bodies (Figs. 4, 5).

Other features of the tumor cells included multiple indentations of the nucleus, rich network of the granular endoplasmic reticulum, well-developed Golgi zones, many pinocytotic vesicles, evenly distributed mitochondria, and a small number of lysosomes. Fre- quently part of the cell surface was covered by inconspicuous basal lamina (Fig. 2). Intercellular connection was uncommon, but desmosome-like struc- tures were found occasionally between tumor cells. Although most of the tumor cells seemed to be myo- fibroblasts, some cells were rather similar to typical fibroblasts, having no bundles of microfilaments. and

2244 CANCER November 15 1980 Vol. 46

FIG. 7. Case 2. Tumor cell culture. Top: Phase contrast photo- micrograph of IDF-2 at the 15th passage ( x 120): Center: Cultured cells have intracytoplasmic inclusions similar to those found in the surgical tissue specimens (H & E, X1.000): Bottom: Acid- phosphatase activity is noted in the cytoplasm of cultured tumor cells. Intracytoplasmic inclusions are seen a s round, pale bodies having no enzymatic activities ( x 1.000).

showing well developed granular endoplasmic reticulum and a considerable amount of 10 nm filaments sparsely distributed in the cytoplasm.

Viral-like particles were never seen in the cytoplasm of cells forming the lesions.

Tissue Culture and Virological Stud-y

Cell lines were established from two patients. They were designated IDF-1 and IDF-2, respectively. Almost all of the IDF-2 cells at the 15th passage level and about 10% of the IDF-1 cells at the 10th passage level contained inclusion bodies which showed the same staining characteristics as those of the tumor cells in the surgical tissue specimens. These bodies had a diameter ranging from I to 12 pm with an aver- age of 3. I pm (Fig. 7).

Electron microscopic study on the cultured cells re- vealed the presence of inclusions similar to those seen in the surgical tissue materials (Figs. 8,9) . Desmosome- like structures were found sometimes between the cells (Fig. 10). Compared with the cells of original tumors, the cultured cells had more abundant lysosomes and numerous microvillous projections, which were incon- spicuous in the original tumor cells (Fig. 8). Particles interpretable as viruses were not seen in the cultured cells. Cocultivations with human embryonic lung cells did not yield any cytopathic changes.

Enzyme Histochemistry

The cytoplasm of most tumor cells in the surgical tissue specimens revealed weak activities of acid phosphatase, a-naphthyl acetate esterase, P-glucuroni- dase, and ATPase, but the cells were evenly negative for naphthol-ASD-acetate esterase, alkaline phos- phatase, and 5'-nucleotidase.

The cells from tissue culture showed moderate to strong activities of acid phosphatase (Fig. 7) and a- naphthyl acetate esterase. The reactions were markedly increased in intensity as compared with those in the surgical tissue specimens. Many cells were weakly positive for P-glucuronidase, but cells with somewhat stronger staining capability were seen also. The ATPase reaction showed a granular activity in the cytoplasm, and a weak activity along the cell mem- brane. The other enzyme reactions were virtually negative in the cultured cells. The intracytoplasmic inclusions seemed to have no obvious activities of the enzymes examined.

Discussion

Recently the concept of myofibroblasts has shed light on the histogenesis of mesenchymal tumors and tumor-li ke conditions. 4,i.lw ' The cells of infantile digital fibromatosis described here may satisfy all of the criteria for the recognition of myofibrobIasts,'8.'1 so that they can be classified as myofibroblasts.

The cytoplasmic filamentous structures are general constituents of many types of cells.'" Based on the morphologic characteristics, they are divided into four

No. 10 INFANTIL E DIGITAL FIBROMATUSIS . I n w s r i h i Pt c i l . 2245

FIG. 8. Case 2. Electron micrograph of the cultured tumor cells showing several inclusion bodies similar to those found in the original tumor cells. The cultured cells have numerous lysosomes and prominent microvillous projections ( ~ 3 , 3 0 0 ) .

main groups: microtubules; 10 nm filaments; micro- filaments of 5 t o 7 nm: and myosin-containing fila- ments. Ishikawa"' suggested that the microfilaments

in nonmuscle cells were actin in nature, and that they were indistinguishable from muscle actin on the mor- phologic o r cytochemical basis.

2246 CANCER November 15 1980 Vol. 46

FIG. 9. Case 2. Detail of the cultured tumor cell containing an inclusion body composed of microfdaments. The surrounding cytoplasm is rich in lysosomes and 10 nm filaments. Many pinocytotic vesicles are seen near the cell membrane ( ~ 2 1 , 2 0 0 ) .

The present study revealed that the intracytoplasmic inclusion bodies of infantile digital fibromatosis were composed chiefly of microfilaments measuring about 5-7 nm. In addition, dense bodies which were similar in appearance to those of the smooth muscle cells were found scattered in the bundles of the microfila- ments extending from the inclusions. These findings suggest that the inclusions may represent a mass of contractile protein composed chiefly of actin filaments. Even at the light microscopic level, the inclusions showed a staining tendency toward the muscular fibrils, staining pink with H & E, bright red with Masson's trichrome, yellow with van Gieson, and purple with PTAH.

On the basis of our present study, we agree with Bhawan et u l . that the inclusion bodies seem to be composed of contractile protein. However, the sig- nificance of myofibroblasts found in digital fibroma- tosis is not clear at this time. Gabbiani and Majno7 suggested that the contraction of myofibroblasts might play a role in the pathogenesis of Dupuytren's contracture. Ryan er ~ 1 . ' ~ proposed that myofibro- blasts were responsible for the contraction of human

granulation tissue. Contracture and deviation de- formity sometimes occurring in infantile digital fibro- matosis may be explained by the action of myofibro- blasts constituting the lesion.

Tissue culture and virologic studies of infantile digital fibromatosis have been undertaken by a num- ber of investigators, but none of them has ever suc- ceeded in demonstrating evident inclusion bodies in the cultured cells.6~13~15~16 I n the present study, cell lines were established from two of the three patients with infantile digital fibromatosis, and the cultured cells proved to contain inclusion bodies obviously showing the same morphologic characteristics as those of the original tumor cells. The result of enzyme histochem- ical studies was not specific, but some lysosomal enzymes were demonstrated in the tumor cells. When compared with the cells of the surgical tissue speci- mens, the cultured cells had more abundant lysosomes and lysosomal enzymes. This phenomenon might have reflected an alteration in cellular metabolism pos- sibly occurring during the process of culture.

Although the inclusion body has been connected with viral etiology of the tumor by some investigators,

No. 10 INFANTILE DIGITAL FIBROMATOSIS . lwasaki ef af. 2247

FIG. 10. Case 2. Parts of two tumor cells from culture showing two desrnosome-like structures between them ( x 3 1,700).

absence of cytopathic effects or virus particles in human embryonic lung cells cocultivated with the tumor cell lines would indicate a nonviral origin of the inclusion body.

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