Intranodal palisaded myofibroblastoma (IPM) is a rare benign spindle-cell tumor of lymph nodes with myofibroblastic/smooth muscle differentiation. We present another case of IPM that confirms the myofibroblastic differentiation of the tumor cells and identifies the so-called amianthoid fibers as collagen deposits by immunohistochemical and ultrastructural techniques. Because IPM shares histomorphologic characteristics with an inflammatory myofibroblastic tumor that has been associated with a virus-induced alteration of cell cycle regulation, the diagnostic approach was extended in this case. We were able to demonstrate cyclin D1 overexpression but could detect neither amplification of the CCND1 gene nor allelic loss at chromosomes 9p22-21 and 13q (surrounding the genes p16 and Rb, respectively). Furthermore, no evidence of human herpesvirus-8 or Epstein-Barr virus infection could be found by polymerase chain reaction or immunostaining. Nevertheless, our results point to the cell cycle regulatory genes as a factor in the pathogenesis of IPM.

Intranodal palisaded myofibroblastoma (IPM) is a rare benign mesenchymal neoplasm of lymph nodes with myofibroblastic/smooth muscle differentiation, often accompanied by the formation of amianthoid fibers.1 More than 40 cases of IPM have been reported in the English language literature since its first description in 1989, and sporadic cases are also known from the non-English language literature.2 

Inflammatory myofibroblastic tumor, another mesenchymal spindle-cell tumor that is also composed of myofibroblasts, was previously associated with Epstein-Barr virus (EBV)3 and human herpesvirus-8 (HHV-8)4 infection. An involvement in the proliferation regulatory pathway of fibroblasts and myofibroblasts has been assigned to DNA sequences and proteins of both HHV-8 and EBV.4,5 Therefore, this case provided us the opportunity to investigate whether IPM could be an additional mesenchymal spindle-cell tumor associated with viral DNA and, similarly, whether there were virus-induced alterations of cell cycle–regulating genes.

A 71-year-old otherwise healthy man presented to the Department of Surgery with a slow-growing tender mass in the left groin. A lymph node was identified and excised to rule out a lymphoma or bacterial infection. The patient was discharged without complication and has been free of disease until now (2 years).

For light microscopy examination, the tissue was fixed in 4.5% phosphate-buffered formalin (pH 7), routinely processed, and embedded in paraffin wax using standard methods. Four-micrometer sections were stained with hematoxylin-eosin, periodic acid–Schiff, van Gieson, Goldner trichrome stain, Weigert resorcin fuchsin for elastic fibers, Gomori impregnation for reticulin fibers, alkaline Congo red for amyloid, and Perls Berlinerblau for hemosiderin.

Immunohistochemical reactions were carried out on formalin-fixed, paraffin-embedded tissue using the streptavidin-biotin-peroxidase method (LSAB-2/HRP system, Dako Corporation, Glostrup, Denmark) with 3,3′-diaminobenzidine-tetrahydrochloride (Dako) as the chromogen (with the exception of cyclin D1: EnVision and Fast-Red). The antibodies used included vimentin, smooth muscle actin (SMA), desmin, S100 protein, cytokeratin (MNF116), epithelial membrane antigen, Ki-67 antigen (MIB-1), CD21, melan A, HMB-45, EBV, p53 (all from Dako), collagen types I, III, and IV (all from Quartett, Berlin, Germany), collagen type II, cyclin D1 (all from Novocastra Laboratories Ltd, Newcastle upon Tyne, United Kingdom), and factor XIIIa (BioGenex, Hamburg, Germany). In addition, an immunohistochemical technique that used sequential co-staining with LSAB/EnVision techniques and 3,3′-diaminobenzidine-tetrahydrochloride and Fast-Red as chromogens was used for SMA and collagen type III. The clones and dilutions are available from us (the authors) on request. The cyclin D1 expression status of the tumor was compared with that of myofibroblastic/fibroblastic areas in 15 nonneoplastic inguinal lymph nodes. As a positive control, nonneoplastic tonsils were used (suprabasal epithelial cells).

The material for ultrastructural examination was initially formalin fixed. The tissue was processed as described previously6 and examined in an EM 910 electron microscope (LEO, Oberkochem, Germany).

Molecular Genetic Analysis

A panel of fluorochrome (6-FAM, HEX, or TAMRA)-labeled polymerase chain reaction (PCR) primer pairs that amplified informative dinucleotide repeat microsatellite loci located on chromosomes 9p (D9S162 and D9S171), 9q21 (D9S197), 13q (D13S217 and D13S263), and 17p (D17S513 and D17S786) was obtained from the Genome Data Base (http://gdbwww.gdb.org). DNA preparation, PCR, and an assessment of the loss of heterozygosity as well as an analysis of cyclin D1 (CCND1) amplification were performed as previously described.6 

HHV-8 DNA analysis was carried out by PCR as reported previously.7 As a positive control, DNA from the cell line CRO-AP3 (DSMZ, Braunschweig, Germany) was used. PCR detection of EBV DNA was performed according to a previous publication.8 The positive control consisted of Raji DNA (provided by U. Wegner). All primer sequences and a detailed description of the PCR reactions are available from us (the authors) on request. To prove the presence of amplificable DNA in the DNA extractions, all of them were amplified with primers for the human beta-globin gene.

Pathologic Findings

Grossly, the excised specimen contained a single lymph node measuring 2.5 cm in its greatest diameter and showing a solid gray-white–to–tan cut surface, a fibrous capsule, and hemorrhagic areas (Figure 1).

Figure 1.

Grossly, the tumor is well circumscribed and has a gray-white cut surface with hemorrhagic areas. Figure 2. Outermost zone of the tumor with sclerosis, hemorrhage, and focal hemosiderin deposits. Extracellular collagenous deposits (amianthoid-like fibers) are evident (hematoxylin-eosin, original magnification ×90). Figure 3. At higher magnification, the amianthoid-like fibers have a deeply eosinophilic core and a lighter periphery. The spindle cells present scant cytoplasm and oval nuclei, occasionally with vague alignment at the fibrillary periphery of the collagenous deposits (hematoxylin-eosin, original magnification ×180). Figure 4. a, Amianthoid-like fibers displaying homogeneous immunostaining for collagen type III (red) and peripheral immunostaining for smooth muscle actin (SMA) (brown) (original magnification ×300). b, The tumor cells show cyclin D1 immunoreactivity (original magnification ×400).

Figure 1.

Grossly, the tumor is well circumscribed and has a gray-white cut surface with hemorrhagic areas. Figure 2. Outermost zone of the tumor with sclerosis, hemorrhage, and focal hemosiderin deposits. Extracellular collagenous deposits (amianthoid-like fibers) are evident (hematoxylin-eosin, original magnification ×90). Figure 3. At higher magnification, the amianthoid-like fibers have a deeply eosinophilic core and a lighter periphery. The spindle cells present scant cytoplasm and oval nuclei, occasionally with vague alignment at the fibrillary periphery of the collagenous deposits (hematoxylin-eosin, original magnification ×180). Figure 4. a, Amianthoid-like fibers displaying homogeneous immunostaining for collagen type III (red) and peripheral immunostaining for smooth muscle actin (SMA) (brown) (original magnification ×300). b, The tumor cells show cyclin D1 immunoreactivity (original magnification ×400).

Close modal

Microscopically, the tumor was composed of variably intersecting bundles of uniform fibroblast-like spindle cells with bland fusiform nuclei and scant eosinophilic cytoplasm. Focal nuclear palisading of the spindle cells, interstitial hemorrhage, and hemosiderin deposits were seen. Mitotic figures were extremely rare. The residual lymph node tissue was compressed to form a small rim on the outermost part of the excision; the inner side of this rim contained a hemorrhagic zone with hemosiderin deposits (Figure 2) and sclerosis. Occasionally, a fibrous pseudocapsule was formed. In addition to the spindle cells, abundant strongly eosinophilic, stellate, extracellular matrix deposits composed of crystalline fibers were seen (Figure 3). These so-called amianthoid fibers stained with van Gieson, Goldner trichrome, and Gomori staining and showed birefringence under polarized light but were negative for Congo red staining and Weigert resorcin fuchsin.

By immunohistochemistry, the amianthoid-like fibers were reactive for collagen types I and III and SMA—the latter especially in their peripheral portions (Figure 4, a). The spindle cells were positive for SMA and vimentin and showed variable immunoreactivity for factor XIIIa. Staining with Ki-67 antigen demonstrated a low proliferative index of less than 5% of the tumor cells. Fifty percent of the tumor cells expressed cyclin D1 (Figure 4, b) (with an appropriate positive control), whereas the percentage of cyclin D1 immunoreactivity of the myofibroblasts/fibroblasts in the nonneoplastic lymph nodes did not exceed 10% (mean index, 7.9%; range, 3%–10%). In all samples, the lymphocytes were negative for cyclin D1 (internal negative control). p53 expression was scattered weakly throughout the tumor. Cytokeratin (MNF116), epithelial membrane antigen, desmin, S100 protein, CD21, collagen types II and IV, HMB-45, melan A, and EBV were negative in the tumor cells.

Ultrastructurally, the spindle tumor cells had long, frequently indented nuclei (Figure 5), pinocytosis vesicles, aggregates of microfilaments, and well-developed rough endoplasmic reticulum with dilated cisternae. The amianthoid-like fibers contained collagen fibrils that were mostly arranged in an orderly parallel fashion with the long axis of the tumor cells (Figure 5).

Figure 5.

Electron micrograph showing a spindle tumor cell with a long, indented nucleus and collagen fibrils from the crystalline deposits (original magnification ×14 000).

Figure 5.

Electron micrograph showing a spindle tumor cell with a long, indented nucleus and collagen fibrils from the crystalline deposits (original magnification ×14 000).

Close modal

Molecular Genetic Analysis

We could not demonstrate a loss of heterozygosity in any of the investigated loci. Neither an amplification nor an allelic loss could be found for the CCND1 gene (cyclin D1).

Despite appropriately reacting positive controls and evidence of amplificable DNA, no HHV-8 or EBV PCR products could be detected in the present case.

The histogenesis of IPM is incompletely understood. The cell of origin is likely the myofibroblast.9 This is supported by the immunophenotype, positive immunostaining for vimentin and SMA, and negative staining for desmin as well as, ultrastructurally, by the evidence of elongated cells with long nuclei and abundant filaments,10 which we could confirm in our case. In accordance with other studies,11 the tumor cells in this case were also positive for factor XIIIa, and the so-called amianthoid fibers were composed of collagen type I- and III-positive collagen fibrils that were arranged from an ultrastructural perspective in mostly parallel fashion to the long axis of the tumor cells.

The following differential diagnoses could be excluded because of the evidence of crystalline extracellular deposits (so-called amianthoid fibers) and criteria as determined in the Table: Kaposi sarcoma, leiomyosarcoma, benign metastasizing leiomyoma, dendritic cell sarcoma, intranodal schwannoma, metastatic spindle-cell lesions (carcinoma as well as malignant melanoma), and inflammatory myofibroblastic tumor.1,10,12 

Differential Diagnoses of Intranodal Palisaded Myofibroblastoma

Differential Diagnoses of Intranodal Palisaded Myofibroblastoma
Differential Diagnoses of Intranodal Palisaded Myofibroblastoma

With regard to the pathogenesis, the predilection of IPM for inguinal lymph nodes is intriguing. After a presentation of IPM in the submandibular region, the previously published inguinal presentations were explained as arising from a sampling problem caused by a limited number of cases.13 Another study found a higher number of myofibroblasts in inguinal lymph nodes than in noninguinal controls, which could be due to the proliferation of myofibroblasts secondary to the increased drainage function in inguinal lymph nodes.14 This result provokes discussion about certain environmental agents or an endogenous (probably genetic) background that could be responsible for the transformation of this preneoplastic hyperplastic status into a neoplasm. Alteration of the 17p locus and p53 protein accumulation seem not to have contributed to the development of the tumor in our case. The cyclin D1 overexpression demonstrated in our case suggests an involvement of cell cycle regulatory genes in the pathogenesis of IPM; however, our loss of heterozygosity data for chromosomes 9p and 13q (all negative) did not point to a pathogenetic role for the p16 and Rb genes (2 integral components of the proliferation regulatory pathway). The immunohistologic finding for cyclin D1 was not accompanied by CCND1 amplification, but previous reports have shown that amplification of CCND1 is not necessarily associated with overexpression of cyclin D1.15 Recently, a correlation of cyclin D1 overexpression with HHV-8 DNA sequences in inflammatory myofibroblastic tumors was reported.4 In addition, this tumor was associated with EBV,3 which contains an oncoprotein (EBNA3C) suggestive of a disruption of multiple cell cycle checkpoints.5 However, in our IPM, there was no evidence for HHV-8 and EBV DNA sequences as demonstrated by PCR, and immunostaining for EBV was also negative. A previous in situ hybridization study of another (recurrent) single IPM also failed to detect a latent EBV infection of the tumor cells.2 

Although a single case report does not allow generalizing conclusions, the cyclin D1 overexpression presented in this study points to the proliferation regulatory pathway as one of the factors involved in the etiologic pathogenesis of IPM. The search for these factors could include viral DNA sequences other than those investigated in this study or additional environmental agents.

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Author notes

Reprints: Britta Kleist, PhD, Institute of Pathology, Ernst-Moritz-Arndt-University, F.-Loeffler-Strasse 23e, D-17489 Greifswald, Germany ([email protected])