Ossifying Fibromyxoid Tumor of the Genitourinary Tract: Report of 4 Molecularly Confirmed Cases of a Diagnostic Pitfall

Abstract

Ossifying fibromyxoid tumors (OFMTs) are rare mesenchymal neoplasms which typically present in the superficial subcutaneous tissues and have not been reported to arise in visceral organs. We now report 4 molecularly confirmed cases of OFMT involving the genitourinary tract. All patients were males, ranging in age from 20 to 66 years (mean 43 years). One case each arose in the kidney, ureter, perirenal soft tissue and penis. All neoplasms demonstrated bland epithelioid to spindled cells set in a variably fibrous to fibromyxoid stroma, and only one had a peripheral shell of lamellar bone. All cases appeared well-circumscribed on gross/radiologic examination, though the primary renal neoplasm permeated between native renal tubules. By immunohistochemistry, S100 protein was negative in all 4 cases, while desmin was positive in two cases. In two cases, the Illumina TruSight RNA Fusion Panel demonstrated a PHF1::TFE3 and EP400::PHF1 fusion, respectively. In the remaining two cases, PHF1 gene rearrangement was confirmed by FISH analysis. Due to unusual clinical presentation, lack of S100 positivity and only occasional bone formation, the correct diagnosis was challenging in the absence of molecular testing. In summary, OFMT may rarely present primarily in the genitourinary tract. Given their nonspecific morphology and immunophenotype, molecular analysis is crucial to establish the correct diagnosis.

Introduction

Ossifying fibromyxoid tumor (OFMT) is a rare mesenchymal neoplasm of uncertain histogenesis^1–5. These neoplasms are typically small in size (median diameter 4 cm) and occur in the subcutaneous tissue or skeletal muscle of the extremities or less often the trunk, in middle-aged adults (median age 49 years, male: female ratio 1.5:1). These neoplasms often display a multinodular architecture, and are comprised of uniform, bland cells which range from epithelioid to ovoid to spindled, arranged in cords and nests in a variably fibrous and fibromyxoid stroma. The amount of fibromyxoid stroma is inversely proportional to the cellularity of the neoplasm. Hyalinization is frequently perivascular. Two-thirds of cases are bounded by a peripheral shell of lamellar bone. The nuclei demonstrate fine chromatin and pinpoint nucleoli, while the cytoplasm is ill-defined. The majority of cases (60–90%) label for S100 protein whereas approximately 50% label for desmin. In addition, cytokeratin is positive in approximately 10% of cases, while immunoreactivity for MUC4, EMA, smooth muscle actin, CD56, and CD10 has also been rarely reported, contributing to diagnostic challenges. Most typical, histologically bland cases carry a low risk of distant metastasis (0–4%). However, cases with marked cytologic atypia, or with a combination of high cellularity and greater than 2 mitoses per 50 high power fields are considered malignant, with a 20–60% risk of metastases¹. Cases with some but not all of these features are defined as atypical OFMT and have a lower risk of metastasis.

OFMTs harbor gene fusions involving the PHD finger protein 1 (PHF1) gene at chromosome 6p21.32 in 50–85% of cases^6–8. The most common fusion involves the EP400 gene at chromosome 12q24.3, which is found in 50% of cases. Other less common PHF1 partners include MEAF6, EPC1, and TFE3^9–11. Identification of characteristic PHF1 gene rearrangements may help expand the existing clinicopathologic spectrum of OFMT. Along these lines, cases originating from the viscera or cases involving the genitourinary tract have not previously been reported.

We report herein 4 cases of genetically-confirmed OFMT which presented as primary genitourinary tract neoplasms. We discuss the broad differential diagnosis such rare neoplasms may trigger.

Methods

Cases

The cases reported herein were retrieved from the consultation files of three authors (PA, BCD, CRA). Cases were identified during a search for potential genitourinary cases among molecularly confirmed OFMT, such as the presence of PHF1 gene rearrangements. Meaningful follow-up is not available for these consultation cases.

Immunohistochemistry

Immunohistochemistry for epithelial markers [cytokeratins AE1/3 and Cam5.2, epithelial membrane antigen (EMA)], neural/melanocytic markers (S100 protein, HMB45, melan A), muscle markers (desmin, smooth muscle actin, caldesmon), CD34, PAX8, Cathepsin K and TFE3 and were performed as previously described¹².

Fluorescence in Situ Hybridization (FISH)

FISH on interphase nuclei from paraffin-embedded 4-micron sections was performed applying custom probes using bacterial artificial chromosomes (BAC). BAC clones were chosen according to UCSC genome browser (http://genome.ucsc.edu)¹³. The BAC clones were obtained from BACPAC sources of Children’s Hospital of Oakland Research Institute (CHORI) (Oakland, CA) (http://bacpacresources.org) and Life Technologies Corporation (Carlsbad, CA). DNA from individual BACs was isolated according to the manufacturer’s instructions, labeled with different fluorochromes in a nick translation reaction, denatured, and hybridized to pretreated slides. Slides were then incubated, washed, and mounted with DAPI in an antifade solution, as previously described¹³. The genomic location of each BAC set was verified by hybridizing them to normal metaphase chromosomes. Two hundred successive nuclei were examined using a Zeiss fluorescence microscope (Zeiss Axioplan, Oberkochen, Germany), controlled by Isis 5 software (Metasystems, Newton, MA). A positive score was interpreted when at least 20% of the nuclei showed a break-apart signal. Nuclei with an incomplete set of signals were omitted from the score.

RNA Sequencing

RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue using Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). Fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-sequencing libraries were prepared using 20 to 100 ng total RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Each sample was subjected to targeted RNA sequencing on an Illumina MiSeq at 8 samples per flow cell (~3 million reads per sample). All reads were independently aligned with STAR (version 2.3) and BowTie2 against the human reference genome (hg19) for Manta-Fusion and TopHat-Fusion analysis, respectively.

Results

Cases and clinical presentation

The neoplasms occurred in 4 males with a mean and median age of 43 years (range, 20–66 years) (Table 1). Case 1 presented as a ureteric mass identified in the workup of unilateral hydronephrosis. Case 2 was a 12 cm renal mass identified on imaging performed for hypertension. Case 3 was a 2 cm enhancing perinephric mass adjacent to the left kidney on imaging, while case 4 was a 2 cm ulcerated penile mass.

Table 1:

Clinical, pathologic and molecular findings of Genitourinary Tract Ossifying Fibromyxoid Tumors (OFMT)

Case	Age/Sex	Clinical Presentation/Site	IHC Positive	IHC Negative	Molecular
1	22/M	Biopsy of a ureteric mass in patient with hydronephrosis	Desmin (patchy)	S100, cytokeratin AE1/3 and 5/6, EMA, PAX8, WT1, smooth muscle actin, CD34, myogenin, TFE3	PHF1 rearranged EP400, EPC1, MEAF6 negative
2	20/M	Nephrectomy for 12cm well-circumscribed renal mass found on imaging performed for hypertension	Vimentin, TLE1, cyclin D1	S100, desmin, EMA, Inhibin, ER, PR, PAX8, WT1, smooth muscle actin, CD34, myogenin, CD117, GATA3, p63, SOX10, STAT6, HMB45, BCOR, SATB2	PHF1 exon 11::TFE3 exon 3 fusion* BCOR, SST FISH negative
3	63/M	Perinephric biopsy of 2cm enhancing soft tissue mass lateral to mid/inferior left kidney	Desmin, SMA, cathepsin K, HMB45 (multifocal), caldesmon (patchy)	S100, cytokeratin AE1/3, S100, CD34, Melan A, CD117, MDM2, CDK4, myogenin, BCOR. H3K27me3 retained. Ki67 near zero	PHF1 rearranged EPC400 negative
4	66/M	Excision of 2cm penile mass		S100, desmin, cytokeratins AE1/3, 34BE12, 8/18, SOX10, , smooth muscle actin, caldesmon, myogenin, CD34, ERG, MDM2,CDK4, ALK, p63. H3K27me3 intact	EP400 exon 37:: PHF1 exon 2 fusion * PHF1 & EP400 rearranged

IHC=immunohistochemistry;

^*tested by Illumina TruSight RNA Fusion panel

Histopathology

All cases demonstrated morphology which, while nonspecific, was compatible with the morphologic spectrum of OFMT. One of the two resected cases (case 2) demonstrated a characteristic peripheral rim of lamellar bone. All neoplasms were composed of bland epithelioid to spindled cells embedded in a fibromyxoid stroma, supported by thin capillary vasculature (Figures 1–4). Case 1 demonstrated predominantly epithelioid morphology and prominent myxoid stroma, while the neoplastic cells in case 2 were predominantly ovoid. Cases 3 and 4 were predominantly spindled with collagenized stroma. Case 2 demonstrated elevated mitotic activity of 2 per 10 high power fields and palisaded tumor necrosis, while case 4 demonstrated an elevated mitotic rate of 3 per 10 high power fields. Thus, both were considered malignant by published criteria. Mitoses were inapparent in cases 1 and 3 in the limited samples obtained by needle biopsy.

Figure 1.

(case 1): This ureteral neoplasm involves the lamina propria and is separated from the benign urothelium (left) by a thin band of hyalinized stroma (A). The neoplasm consists of cords of bland epithelioid cells set within a predominantly myxoid stroma which is accentuated around small capillaries (B). High power view demonstrates the bland cytology of the neoplastic epithelioid cells (C). The neoplastic cells demonstrate patchy immunolabeling for desmin (D).

Figure 4.

(case 4): At low power, this penile lesion is centered in the subepithelial stroma, and separated from the overlying squamous mucosa at the left by a thin band of fibrous tissue (A). Focally, the neoplasm ulcerates the squamous mucosa (B). The neoplasm is predominantly spindled, with mainly fibrous and focally myxoid stroma. Perivascular hyalinization is present (C). At high power, the neoplastic cells are spindled and bland cytologically. Mitotic activity is evident (D).

In limited biopsy material, case 1 appeared well-delineated and separated from the overlying renal pelvic urothelium by a band of hyalinized fibrous tissue. Similarly, case 4 appeared largely well-delineated and separated from the overlying penile squamous mucosa by hyalinized fibrous tissue, though the neoplasm focally ulcerated the mucosa. While case 2 appeared well-circumscribed grossly and on imaging, the neoplastic spindled cells encircled native renal tubules which were scattered throughout the lesion.

The neoplasms demonstrated a nonspecific immunohistochemical profile (Table 1). Notably, all were negative for S100 protein and only two of four labeled for desmin. Case 3 demonstrated patchy immunoreactivity for HMB45 and cathepsin K. All cases were negative for epithelial markers, myogenin and CD34.

Molecular Analysis

All 4 cases demonstrated evidence of PHF1 gene rearrangement. Two cases were tested by targeted RNA sequencing: case 2 demonstrated a PHF1::TFE3 fusion (PHF1 exon 11 fused to TFE3 exon 3), while case 4 harbored an EP400::PHF1 fusion (EP400 exon 37 fused to PHF1 exon 2). The latter case was further confirmed by FISH showing both PHF1 and EPC400 gene rearrangements, respectively. Cases 1 and 3 showed evidence of PHF1 gene rearrangement by FISH though a partner could not be identified (Table 1, Figure 5).

Figure 5.

FISH analysis showing the presence of PHF1 (case 3) (A) and TFE3 (case 2) (B) gene rearrangements showing a break-apart signal pattern (red, centromeric; green, telomeric).

Discussion

The genetic hallmarks of ossifying fibromyxoid tumor (OFMT) are gene fusions involving the PHD finger protein 1 (PHF1) gene at chromosome 6p21.32, which are found in 50–85% of cases^6–8. PHF1 protein regulates expression of developmental genes by interaction with polycomb-repressive complex 2 (PRC2), resulting in alterations in chromatin structure. The most common fusion involves the EP400 gene at chromosome 12q24.3, which is found in 50% of cases. Other less common PHF1 partners include MEAF6, EPC1, and TFE3^9–11. Rare fusion variants such as KDM2A::WWTR1, CREBB::BCORL1, and ZC3H7B::BCOR have also been reported¹⁴. In approximately 15% of cases, PHF1 is known to be rearranged but a fusion partner has not been identified. These cases have similar clinicopathologic features as those in which a PHF1 fusion partner has been identified. Several of the fusions identified in OFMT, including MEAF6::PHF1, EPC1::PHF1, and ZC3H7B::BCOR, have also been reported in endometrial stromal sarcoma^15–17. The less common fusions in OFMT, particularly the PHF1::TFE3 fusion, may correlate with a more aggressive behavior and less frequent immunoreactivity for S100 protein^8,9.

We report 4 molecularly confirmed cases of OFMT arising primarily in the genitourinary tract. OFMT typically arises in the superficial soft tissue of the extremities or trunk. To our knowledge, examples of this neoplasm presenting primarily within visceral organs (such as cases 1 and 2 of this series) have not been reported. Two cases of OFMT were previously reported in the retroperitoneum and retrorectal soft tissue^{18, 19} , though these were not molecularly confirmed and did not involve viscera Schoolmeester et al. reported an ossifying cardiac sarcoma with a JAZF1::PHF1 fusion which raised the possibility of malignant OFMT, though as the authors noted the morphology was beyond that accepted for malignant OFMT²⁰. Hence, OFMT would likely not be considered highly in the differential diagnosis of these neoplasms given their relatively nonspecific monomorphic epithelioid to spindle cell morphology and immunophenotype. While most classic OFMTs have a consistent immunoprofile with S100 protein positivity with or without desmin staining, the cases in our series lacked this pattern. Notably, all cases were negative for S100 protein, with 2 of 4 cases demonstrating immunoreactivity for desmin. One case demonstrated immunoreactivity for HMB45 and cathepsin K which has not previously been reported in OFMT. Some reported OFMT, specifically those with the PHF1-TFE3 fusion, have labelled diffusely for cytokeratin and neuroendocrine markers such as synaptophysin, causing confusion with metastatic neuroendocrine carcinoma⁹. The nonspecific morphology and potentially misleading immunophenotype highlights the crucial role of molecular pathology in establishing the diagnosis of this rare neoplasm in these unusual sites.

OFMT of the genitourinary tract raises a broad differential diagnosis of other uncommon but well described entities. Some of these are specific to the genitourinary tract while others are soft tissue neoplasms not limited to this site. The more epithelioid, nested appearance of the ureteral tumor in case 1 raised the differential diagnoses of nested urothelial carcinoma. The latter is a particularly difficult diagnosis to make in the upper urinary tract, given the often florid nature of von Brunn nests in this site. The absence of immunoreactivity for cytokeratin helped exclude that possibility²¹. The bland cytology and relatively even distribution of native renal tubules within the lesion in case 2 raised the differential diagnosis of a mixed epithelial stromal tumor (MEST)²². However, the absence of immunoreactivity for estrogen and progesterone receptors, as well as the absence of characteristic epithelial patterns of MEST (cysts, intracanalicular growth, etc.) argued against that possibility. Perivascular epithelioid cell tumor (PEComa) was another consideration, given particularly in case 3, which demonstrated immunoreactivity for HMB45 and cathepsin K. PEComas characteristically demonstrate TSC1/2 mutations or TFE3 gene fusions, and do not demonstrate PHF1 rearrangements^23–25. GLI1-rearranged or amplified tumors have a similar bland morphology and fibromyxoid stroma as does OFMT, and have recently been described in the genitourinary tract^26,27. Like OFMT, GLI1 altered neoplasms have a variable immunophenotype, with S100 and actin being the most commonly expressed markers, though these certainly are not found in each case. The extent of morphologic and immunohistochemical overlap between OFMT and GLI1-altered neoplasms is so substantial that molecular testing is essential to definitively distinguish between these two entities. Myoepithelial neoplasms also may be considered in the differential and may rarely present in the genitourinary tract. Such neoplasms share with OFMT S100 immunoreactivity, fibromyxoid stroma and typically bland cytology. However, myoepithelial tumors consistently express epithelial markers and have a high prevalence of EWRS1 or FUS gene rearrangements, but not PHF1 gene rearrangement^28,29. Low grade fibromyxoid sarcoma/sclerosing epithelioid fibrosarcoma (LGFMS/SEF) is another consideration and has been reported to occur in the genitourinary tract^30,31. Of note, OFMT may label for the LGFMS/SEF marker MUC4, though rarely and only focally³². However, LGFMS/SEF show pathognomonic FUS and EWSR1 related gene fusions^33–35. The combination of bland ovoid cells and capillary vasculature may raise in addition the possibility of either a BCOR-altered sarcoma or a solitary fibrous tumor, both of which may present in the genitourinary tract^36,37. The absence of BCOR immunoreactivity (demonstrated in the two cases tested in this series) would argue against these entities. Extraskeletal myxoid chondrosarcoma also share smonotonous round cell cytology, myxoid stroma and nested architecture with OFMT, but typically lacks S100 protein staining and demonstrate rearrangements of the NR4A3 gene^38,39.

In summary, we report four cases of OFMT arising in the genitourinary tract. These cases expand the clinicopathologic spectrum of OFMT to include presentation within visceral organs, and highlight the critical role of molecular pathology in diagnosis.

Figure 2.

(case 2): At first glance, the neoplasm initially appears well-delineated from the kidney (bottom), however, native renal tubules are present within the neoplasm (A). At its periphery, an incomplete shell of lamellar bone is present (B). The neoplasm demonstrates areas of palisaded necrosis (C). Focal areas demonstrated concentric growth around intratumoral capillaries (D). The neoplasm also encircles native renal tubules within the lesion in a concentric fashion (E). At high power, the neoplastic cells demonstrate bland ovoid nuclei and ill-defined pale cytoplasm, with scattered mitotic figures (F).

Figure 3.

(case 3): The needle core biopsy of this perirenal mass demonstrates a bland spindle cell neoplasm in fibrous stroma (A, B). Mitotic figures are not apparent. The neoplastic cells demonstrate patchy labeling for caldesmon (C) and HMB45 (D).