J Clin Pathol 1991;44:761-764 761 Selective location of palisaded myofibroblastoma with amianthoid fibres J Clin Pathol: first published as 10.1136/jcp.44.9.761 on 1 September 1991. Downloaded from

G Bigotti, A Coli, M Mottolese, F Di Filippo

Abstract Methods The occurrence of a palisaded myo- Tumour specimens were fixed in 10% for- fibroblastoma with amianthoid fibres malin and embedded in paraffin wax. in the left inguinal of a 51 Hematoxylin and eosin stained sections were year old man prompted an investigation examined by light microscopy to establish the of the factors underlying its exclusive diagnosis. Tissue sections were also stained location. The antigen profile was charac- with Masson's trichrome, Van Gieson stain, ternsed which confirmed the homogen- Gomori's methenamine silver, reticulin stain, eous expression of vimentin and smooth Congo red, Giemsa and methylene blue. muscle actin as well as the lack of des- Ten to 15 tumour free lymph nodes from min. Use of monoclonal antibodies to each of lateral cervical, axillary, mediastinal, check for a differential distribution of abdominal, lumboaortic, pelvic and inguinal myofibroblasts and the putative cell of regions were obtained from patients under- origin of palisaded myofibroblastoma going surgery, primarily for . Tissue showed that inguinal lymph nodes have specimens were routinely processed and abundant vimentin and actin positive stained for histopathological diagnosis. cells and desmin negative cells. This sug- Giemsa and methylene blue stains were used gests that the selective occurrence of to evaluate the mast cell content. For myofibroblastoma is related to the nodal immunohistochemistry 4 gm thick tissue sec- microenvironment, providing a source tions from both the tumour and the various of available and potentially proliferating lymph nodes, chosen as previously described, myofibroblasts. Mast cells abounded in were dewaxed in xylene and hydrated in this lesion, particularly around amianth- graded ethanol; endogenous peroxidase oid fibres, as well as in pelvic and activity was blocked with methanolic inguinal lymph nodes. hydrogen peroxide and slides were sub- In view of the known role of mast cells sequently washed in phosphate buffered saline in interstitial matrix degradation it is (PBS). The sections were then processed for http://jcp.bmj.com/ postulated that the core of amianthoid immunoperoxidase according to the method fibres represents degraded interstitial of Hsu et al.4 matrix, analogous to the sclerotic areas The reagents were purchased from Vector commonly found in the above mentioned Laboratories (Vectastain, Burlingame, lymph node groups, while the peripheral California, USA). The staining reaction was spokes, so peculiar to this entity, are the developed with 3,3-diaminobenzidine tetra- result of vimentin and smooth muscle hydrochloride. Sequential sections from each on September 29, 2021 by guest. Protected copyright. actin, directly shed by -proliferating of the lymph node blocks-that is, sections of myofibroblasts. tissue with virtually identical architecture- were examined to compare their immunoreac- tivity after staining with monoclonal Primary tumours of the lymph nodes, exclud- antibodies. The following antibodies were ing those of lymphoid origin, are rare. Apart used at dilutions suggested by the manufac- from Kaposi's which occurs in those turer: S100 protein (Dako); vimentin (Dako); with AIDS,' melanocytic nevi, and pseudo- smooth muscle actin (Ortho); desmin (Dako); sarcomatous malignant from dis- and factor VIII (Dako). S100 and factor VIII Department of placed nevic cells,2 other primary lymph node antobodies were applied only to sections of Pathology, Catholic tumours are uncommon. Secondary spindle tumour. University of Sacred cell tumours are represented by malignant Negative controls included tissue section , Largo F. Vito 1, 00168 Rome, Italy melanomas, pseudosarcomatous ,3 incubation with normal horse serum or PBS G Bigotti and metastatic . We report the buffer instead of the primary antibody. A Coli occurrence of a primary spindle cell tumour in Positive controls consisted of normal intes- Department of an inguinal lymph node with the classic tinal wall, and malignant ; they dis- Pathology and features of the so-called "palisaded myo- and Surgery, Regina Elena played strong specific immunorectivity. Cancer Institute, fibroblastoma with amianthoid fibres." We After being stained with Giemsa and Rome attempt to explain its selective occurrence in methylene blue immunohistochemically M Mottolese inguinal lymph nodes by investigating the stained cells and metachromatic mast cells F Di Filippo differential myofibroblastic content of lymph were counted in each of 20 randomly selected Correspondence to: x Giulio Bigotti nodes of various sites. We also try to explain fields using a medium power objective ( 250) Accepted for publication the histogenesis of the amianthoid fibres and a field average was calculated for each of 4 April 1991 typical of this entity. the different lymph node groups. 762 Bigotti, Coli, Mottolese, Di Filippo

Case report proliferation of spindle cells with elongated, A 51 year old man presented with a swelling tapered nuclei and weakly staining eosino-

of the left inguinal region of two years dura- philic cytoplasm lacking distinct fibrils (fig 1). J Clin Pathol: first published as 10.1136/jcp.44.9.761 on 1 September 1991. Downloaded from tion and recent increase in size. A lymph The tumour cells were arranged in bundles node, measuring 2 x 3 cm, was recovered at sometimes intersecting at acute angles and in surgery. Fine needle aspiration biopsy had led some areas showing distinct palisading of the to a cytological diagnosis consistent with sch- nuclei. Throughout the tumour there were wannoma. A frozen section was cut and inter- multiple collections of an intensely eosino- preted as haemorrhagic infarct of the lymph philic collagen matrix which were generally node. On cut section the lymph node seemed oval in shape and had peripheral spokes to have been replaced by grey-white tissue strictly contiguous with proliferating cells. In with a prominent rim of blood at the peri- a few areas these collections of eosinophilic phery and flecks of bleeding in the tumour matrix seemed to be centered around blood tissue itself. vessels (fig 2). The tumour was circumscribed The patient remained well with no by a thick collagenous capsule and beyond the recurrence one year after excision. capsule remnants of lymph node with mar- ginal sinuses were found. The tumour was also haemorrhagic, and in many areas red Results blood cells were seen within the interstitium MYOFIBROBLASTOMA among proliferating cells. Less than two Histological examination of formalin fixed, mitoses per 50 high power fields were found. paraffin wax embedded tumour showed a Trichrome and Van Gieson stains did not show distinct longitudinal fibrils in tumour cells and the amianthoid fibres had the features of strongly stained collagen at their cores- blue with Trichrome and red with Van Gieson (fig 3). Gomori's stain gave the amian- thoid fibres a strong black colour; they were Congo red stain positive and appeared bire- fringent under polarised light. Giemsa and methylene blue stains showed numerous mast cells with metachromatic granules, which clustered around the amianthoid fibres (fig 4) and which were diffusely scattered far away from the latter in areas of active cellular proliferation. Immunohistochemistry showed tumour cell staining for vimentin and smooth muscle actin

(fig 5), while immunostaining for desmin, fac- http://jcp.bmj.com/ tor VIII, and S100 was consistently negative. The positivity in all cases was cytoplasmic. The amianthoid fibres were negative for all the immunocytochemical markers except in the spokes contiguous with cell proliferation Figure I Palisaded myofibroblastoma showingfascicles ofelongated cells with tapered where positivity for vimentin and actin could nuclei andpale cytoplasm. be observed (fig 5). S100 and antifactor VIII on September 29, 2021 by guest. Protected copyright. antibodies stained, respectively, only rare nerve bundles and vascular endothelia.

LYMPH NODES Giemsa and methylene blue stains showed numerous mast cells, especially around areas of sclerosis in pelvic, inguinal, and lumboaor- tic lymph nodes. By contrast, mast cells were scarce and isolated in other lymph node

* ~ -' groups. In all the locations examined mast 4C *¾'-A ' ^ >t -'s4; ,.^,9. *; ~~.5k^i ;.* i~.* ;X 0 cells had a preferential distribution in lymph 4~ ~~~~~~4. sinuses, toward the medulla, and around areas of sclerosis where this was present. Immunostaining of lateral cervical, axillary, mediastinal, abdominal, lumboaortic and pelvic lymph nodes highlighted a striking paucity of smooth muscle actin, vimentin 4.. 144 positive, desmin negative elongated cells (fig -* ; e . *; vs t- w - n 6); these were identified mainly in the capsule and fibrous septa. Smooth muscle, stained by monoclonal antibodies to vimentin, smooth muscle actin, and desmin, was present in vessel walls and bundles of elongated cells in Figure 2 Section of an early phase of amnanthoidfibre formation, in which a vessel is the capsule, and in lesser numbers in fibrous beginning to be surrounded by collagen. Overlaying is an almost fully formed amianthoid structure. Both seem to be surrounded by proliferating myofibroblasts. trabeculae. Occasional, rare, and isolated des- Selective location ofpalisaded myofibroblastoma with amianthoidfibres 763 J Clin Pathol: first published as 10.1136/jcp.44.9.761 on 1 September 1991. Downloaded from

Figure 6 Smooth muscle actin antibody stains only the -* .-7A q _ -- 5 vessel walls in this abdominal lymph node. Note the Figure 3 The so-called "amianthoidfibres" seem to be composed of a central collagen virtual absence of immunostained interstitial cells. core and peripheral spokes, as shown here. Note that this structure is strongly stained by Van Gieson and is centered by a blood vessel. :~~

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- I ,z . -s ._5I -e L .t.-_ : --O I"7..-1_, Figure 7 Section of inguinal lymph node showing numerous smooth muscle actin positive interstitial cells interdigitating with lymphocytes. sometimes of reticular min positive cells, on September 29, 2021 by guest. Protected copyright. were seen between lymphocytes. Figure 4 Numerous methylene blue stained mast cells are seen around amianthoidfibres shape, and among proliferating tumour cells. In contrast, inguinal lymph nodes had large numbers of elongated, vimentin and actin positive, desmin negative cells, which were widespread in the capsule, fibrous septa, and interdigitating with the lymphoid tissue (fig 7). ~~ ~ ~ ~ ~ ~ ~ ~ ~ Their distribution was predominantly subcap- sular, paracortical, sinusoidal and toward the medulla, and virtually never in follicles and in particular not in germinal centres. These cells seemed particularly abundant and clustered around areas of sclerosis. Their immunocyto- chemical staining was cytoplasmic. Inguinal lymph nodes, stained with the des- min monoclonal antibody, had an immuno- reactivity pattern similar to that of other lymph node regions, with only rare and VW ~ ~V isolated stained cells, identified mainly in the subcapsular and paracortical areas. The table summarises the mast cell count and the actin and desmin immunoreactivity in the different lymph node groups.

A.,~~~ ~ ~ ~ ~ ~ ~ ~ ~ Discussion We have reported a rare entity56 which is Section myofibroblastoma showing dark staining smooth muscle Figure of palisaded to in lymph actin positive tumour cells and spokes of amianthoid fibres (arrows). thought arise selectively inguinal 764 Bigotti, Coli, Mottolese, Di Filippo

Table Immunoreactive cells and mast cell detection in various lymph node sites characterised by a variable percentage of des- No of min expression, should nevertheless show a

Case No lymph nodes Actin Desmin Mast cell count reactivity of at least 50%." J Clin Pathol: first published as 10.1136/jcp.44.9.761 on 1 September 1991. Downloaded from I inguinal 15 + + -to+/ + + We feel that mast cells may have a crucial 2 inguinal 10 + + - to+/- + + role, both in the formation ofamianthoid fibres 3 iliac 10 + - + and in the active proliferation of 4 iliac 13 - to/ - + + myofibroblasts 5iliac 12 +/- +/- -to +/- in myofibroblastoma. In fact, mast cells have 6 obturator 12 + + + + 7 aortocaval 14 + +/- + + been shown to be involved in interstitial matrix 8 peripancreatic 10 -to + /- to + / degradation and to be potent stimulators of 9 perigatric 13 10 mediastinal 10 -to + - to + to + - fibroblasts and of tumour cell proliferation, an 11 axillary 12 + - to+ - action mediated by histamine and heparin 12 axillary 13 - to +/ 13 lateral-cervical 11 release.'3 '4 In our study we have shown a differential Key: immunostained cells present only in the capsule and thick septa. One or no mast cells per distribution of mast cells medium power field (MPF); + /- = immunostained cells in the lymphoid tissue; one or no according to the stained cells per MPF, two to four mast cells per MPF; + = few to moderate immunostained lymph node region and their preferential loca- cells in the lymphoid tissue, two to 10 stained cells per MPF, five to 10 mast cells per MPF; + + = numerous immunostained cells in the lymphoid tissue, 11 or more stained cells per tion in sinuses and around areas ofsclerosis, the MPF, 11 or more mast cells per MPF; -to + / = average obtained from two contiguous latter commonly found in otherwise normal values. Note. Capsular and trabecular immunostaining was observed in all the lymph nodes examined. inguinal and pelvic nodes; in this context mast cells could be responsible for sclerosis by nodes. Its recognition is of great importance releasing protease in the intercellular matrix because ofits possible confusion with a primary and around blood vessels. malignant or metastatic spindle cell tumour. The same mechanism could operate in the Palisaded myofibroblastoma, a term firstused production ofthe collagen core of the amianth- by Weiss et al,5 can closely simulate Kaposi's oidfibresofpalisadedmyofibroblastoma. Suster sarcoma, which in patients with AIDS can et al6 showed that it is composed of degraded occur as a primary in lymph nodes; this is collagen. The characteristic peripheral spokes, particularly evident when the interstitial composed of vimentin and smooth muscle haemorrhage resembles the extravasated red actin, would then probably represent inter- blood cells seen in Kaposi's sarcoma. The lack mediate filaments directly extruded from the of true vascular slits and nuclear atypia, as well massively proliferating myofibroblasts at the as to the extreme rarity of mitotic figures, will interface with such a core. usually indicate the correct diagnosis. Further- The recognition of palisaded myofibroblas- more, Kaposi's sarcoma has a sinusal distribu- toma remains very important, especially in tion, and when lymphoid tissue is present it is view of the uniformly favourable prognosis always located deep within the tumour and not (lack ofrecurrence, local invasion, and systemic at the periphery, as seen in myofibroblastoma.7 spread) as seen in our case as well as in the The histogenesis of this entity is not com- others previously reported. pletely understood but it is suggested that the We are grateful to T Prosperi and A for

Ruggeri excellent http://jcp.bmj.com/ cell of origin is the myofibroblast,5 first des- technical assistance and to G Rosi, R Mallucci, and B Ghirotti cribed by Gabbiani et al in granulation tissue as for photographic assistance. well as in quasineoplastic and neoplastic condi- 1 Finkheiner WE, Egberst BM, Groundwater JR, Sagabiel tions such as in various types of fibromatosis.9 RW. Kaposi's sarcoma in young homosexual men. A histologic study with particular reference to lymph node It displays a generally distinct antigenic profile involvement. Arch Pathol Lab Med 1982;106;261-4. that is characterised by the expression of 2 Azzopardi JG, Ross CMD, Frizzera G. Blue nevi of lymph node capsule. Histopathology 1977;1:437-61. vimentin, smooth muscle actin, procollagen 3 Leifer C, Miller AS, Putong PB, Min BH. Spindle cell type I, III, and V;'° " more recently, however, of the oral mucosa. A light and electron on September 29, 2021 by guest. Protected copyright. microscopic study of apparent sarcomatous metastasis to the existence of different phenotypic subsets of cervical lymph nodes. Cancer 1974;34:597-605. myofibroblasts has been pointed out.'2 Pali- 4 Hsu S-M, Raine L, Fanger H. Use of avidin-biotin-perox- idase complex (ABC) in immunoperoxidase technique. A saded myofibroblastoma is characterised by the comparison between ABC and unlabeled antibody (PAP) continuous expression of actin and vimentin procedures. J Histochem Cytochem 1981 ;29:577-80. 5 Weiss SW, Gnepp DR, Bratthauer GL. Palisaded and by the lack of desmin,5 as seen in our case. myofibroblastoma. A benign mesenchymal tumor of Its selective occurrence has prompted us to lymph node. Am J Surg Pathol 1989;13:341-6. 6 Suster S, Rosai J. Intranodal hemorrhagic spindle-cell investigate the possibility of a differential tumor with "amianthoid" fibers. Report of six cases of a myofibroblastic distribution according to the distinctive mesenchymal of the inguinal region that simulates Kaposi's sarcoma. Am J Surg Pathol lymph node site. 1989;13:347-57. Our results indicate that actin positive, des- 7 Amazon K, Rywlin AM. Lymph node involvement in Kaposi's sarcoma. Am J Dermatopathol 1979;1: 173-6. min negative myofibroblasts are strikingly 8 Gabbiani G, Ryan GB, Majno G. Presence of modified prominent in inguinal lymph nodes where they fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 1971;27:549-50. are diffusely present as opposed to other lymph 9 Gabbiani G, Majno G. Dupuytren's contracture: fibroblast node chains. In this context palisaded contraction? An ultrastructural study. Am J Pathol 1972,66:131-46. myofibroblastoma could represent a quasi- 10 Oda D, Gown AM, Vande Berg JS, Stern R. The fibroblast- neoplastic or neoplastic proliferation in a group like nature of myofibroblasts. Exp Mol Pathol 1988; 49:316-29. of lymph nodes subject to a striking degree of 11 Schurch W, Semayer TA, Lagace R, Gabbiani G. The drainage function and massive workload. intermediate filament cytoskeleton of myofibroblasts: an immunoflourescence and ultrastructural study. Virchows The numerous vimentin positive, smooth Arch (Pathol Anat) 1984;403:323-36. muscle actin positive, desmin negative cells we 12 Skalli 0, Schurch W, SeemayerT, etal. Myofibroblasts from diverse pathologic settings are heterogeneous in their have described in the inguinal lymph nodes content of actin isoform and intermediate filament could be smooth muscle cells and not proteins. Lab Invest 1989;62:275-85. 13 Levi-Schaffer F, Kupietzky A. Mastcells enhance migration myofibroblasts, but in this instance one would and proliferation offibroblasts into an in vitro wound. Exp have expected to find a much higher percentage Cell Res 1990;188:42-9. 14 Roche WR. Mastcellsandtumors.Thespecificenhancement of reactivity with the desmin monoclonal of tumor proliferation in vitro. Am J Pathol 1985;119: antibody because smooth muscle cells, though 57-64.