Aims Angiomatoid fibrous histiocytoma (AFH) is histologically typified by nodules of histiocytoid spindle cells with pseudoangiomatoid spaces, fibrous pseudocapsules and lymphocytic cuffs. The principal goal was to expand the spectrum of AFHs through clinicopathological and molecular characterisation.
Methods Thirteen AFHs, including 11 with confirmed hallmark translocation, were reappraised for classic features, reactive osteoclasts, mitoses and stromal, architectural and cytomorphological variations, with CD99, desmin and EMA stained in available cases.
Results Seven male and six female patients ranged in age from 4 to 63 years (median, 13), including 4 older than 20 years. Tumours were located on the extremities (n=6), trunk (n=4) and scalp (n=3). Although fibrous pseudocapsules were observed in all cases, four showed solid histology without pseudoangiomatoid spaces and another one lacked peripheral lymphoid infiltrates. Nuclear pleomorphism was striking in two cases, moderate in seven and absent in four, with osteoclasts seen in two cases. In three AFHs with sclerotic matrix, one exhibited perivascular hyalinisation and nuclear palisading, reminiscent of a schwannoma. In three varyingly myxoid tumours, one closely resembled a myoepithelioma with prominent reticular arrangement of spindle cells in an abundant myxoid stroma. Besides EWSR1 gene rearrangement detected in four cases by fluorescence in situ hybridisation (FISH), EWSR1-CREB1 fusion was confirmed in nine cases, including a schwannoma-like AFH, and EWSR1-ATF1 fusion detected in a myoepithelioma-like AFH. Immunohistochemically, 56% of AFHs were positive for EMA, 78% for desmin and 100% for CD99.
Conclusions Molecular testing is diagnostic of variant AFHs displaying diverse histomorphological alterations in the architectural patterns, cytomorphology and extracellular matrix.
- SOFT TISSUE TUMOURS
- MOLECULAR PATHOLOGY
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Angiomatoid fibrous histiocytoma (AFH) is a slow-growing, rarely metastasising mesenchymal neoplasm of uncertain differentiation and preferentially affects children and young adults.1 ,2 Histologically, typical AFHs are characterised by multinodular proliferation of oval histiocytoid to spindle cells with syncytial growth, forming sheets, whorls or vague bundles, and accompanied by a triad of central pseudoangiomatous spaces, a thick fibrous pseudocapsule and pericapsular lymphoplasmacytic cuffing in varying proportions.1 ,2 The diagnosis of AFH is straightforward when it displays a classical histology. However, AFH may rarely exhibit unusual clinicopathological features, such as older ages at presentation, occurrence outside somatic soft tissues and alterations in the architectural patterns, stromal matrix and cytomorphology.1–4 Especially in the absence of the classical triad, these histomorphological variations may pose a great diagnostic challenge. This is further complicated by the lack of diagnostic markers for AFH, although Epithelial Membrane Antigen (EMA), desmin and CD99 are variably positive in approximately two-thirds of cases.1 ,2
The molecular pathogenesis of AFH has become clarified by the recent discovery of hallmark fusion genes resulting from various chromosomal translocations, including the most prevalent EWSR1-CREB1 derived from t(2;22)(q33;q12),3 ,5 followed by EWSR1-ATF1 from t(12;22)(q13;q12)6 ,7 and the least common FUS-ATF1 from t(12;16)(q13;p11).8 Therefore, molecular testing is of great diagnostic utility for AFHs, especially in the case of atypical clinicopathological presentation. In this series, we have expanded the clinicopathological spectrum of AFH involving superficial soft tissues by characterising 13 cases with unusual histomorphology at variable degrees, including 11 with molecular confirmation by fluorescence in situ hybridisation (FISH) and/or reverse transcription-polymerase chain reaction (RT-PCR).
Materials and methods
This retrospective study was approved by the Institutional Review Boards of Taipei Medical University (201208040) and Chang Gung Memorial Hospital (101-3392B). AFHs confirmed by molecular testing were retrieved from the consultation file (H-YH), yielding 10 cases with haematoxylin/eosin (HE) and immunohistochemical stains at time of diagnosis for further analysis of the clinicopathological features. The pathology archives of participating institutes were sought for cases coded as ‘fibrous histiocytoma’ with the following descriptors, such as angiomatoid, aneurysmal, syncytial or solid growth and lymphoid cells. According to the 2013 WHO Tumour Classification,1 three potential AFH cases with available formalin-fixed tissues were identified for additional immunostaining and molecular testing. A total of 13 cases formed the basis of this study on clinicopathological and molecular characterisation of AFH, with special attention to the spectrum of variant histomorphology.
The clinical information, treatment and follow-up data were obtained from the referring pathologists and/or treating physicians. HE sections were reviewed by three authors (YCK, JL, H-YH) for each case to record various histological features, including the presence or absence of pseudoangiomatous spaces, fibrous pseudocapsules and lymphocytic cuffs, mitoses per 10 HPFs (1 HPF=0.196 mm2) and the spectra of growth pattern, extracellular stroma and cytomorphological pleomorphism. The architecture of tumour growth was classified as predominantly solid, multinodular or reticular with or without focal nesting or palisading arrangement. The alterations of stromal matrix were categorised as hyaline/sclerosing, myxoid/fibromyxoid or entirely syncytial growth without apparent matrix deposition or degeneration. Besides typical histiocytoid or spindle cells, variant pleomorphic or small blue tumour cells as well as reactive osteoclast-like giant cells were assessed for their presence and distribution. The degree of nuclear pleomorphism was graded as striking, moderate or absent.
Immunohistochemistry for desmin, EMA and CD99 was performed and scored as detailed in the online supplementary data.
Two aneurysmal fibrous histiocytomas, easily confused with AFH in both nomenclature and histology, served as negative controls throughout to ensure technical reliability. The primers, probes, procedures and interpretations of FISH and RT-PCR are provided in the online supplementary data.
The salient clinicopathological, immunohistochemical and molecular findings are tabulated in table 1.
Clinical findings and follow-up data
The 13 patients with AFH comprised seven males and six females and ranged in age from 4 to 63 years (mean 20.8, median 13), with four patients being older than 20 years at diagnosis. The majority of cases were described as a slowly-growing, superficial painless nodule or mass without antecedent traumatic history or constitutional symptoms. Tumours were located on the lower limbs in four cases, upper limbs in two, trunk in four and scalp in three. Simple excision was performed in all patients without adjuvant radiotherapy or chemotherapy. One patient (case 11) initially received wide excision but was afflicted with sequelae of limb deformity and limitation in motion. Follow-up was available in nine patients, and all were alive without recurrence or metastasis after a median duration of 24 months (range 1–108).
Grossly, all tumours were circumscribed in the outer surfaces and ranged from 0.6 to 5.5 cm (mean 2.3, median 2) in the greatest dimension. Red-brown haemorrhagic cystic spaces of variable sizes were described in nine cases but absent in the remaining four. Among the latter group, one AFH (case 9) displayed prominent myxoid change with gelatinous consistency.
Histologically, nine cases were entirely confined to the subcutis (figure 1A), three centred between the dermis and subcutis and one predominantly located in the subcutis with focal minimal involvement of underlying skeletal muscle. In each case, a mononodular or multinodular proliferation of oval histiocytoid to spindly tumour cells with vesicular nuclei and indistinct cell boundaries was observed (figure 1A,B), which assumed at least focally a syncytial whirling or sheet-like growth pattern of classical AFHs (figure 1A,B). Pseudoangiomatoid spaces of variable sizes were seen in nine cases, while the remaining four cases were completely devoid of such haemorrhagic pools (figure 1A). In contrast, fibrous pseudocapsules, albeit not necessarily completely encircling the lesion, could be discerned at least focally. Notably, there was only a single AFH arising from the scalp that lacked lymphoid cuffing (case 2). Taken together, all 13 cases in this study showed at least two features of the classical histological triad. Three cases (4, 5 and 10) showed focal to prominent sclerosing changes in the stromal component (figure 1C), another four cases displayed either a fibromyxoid (figure 1D, cases1, 7 and 13) or predominantly myxoid (figure 2A, case 9) stromal matrix and there was no remarkable stromal alteration in the remaining six cases. The tumour cells of the predominantly myxoid AFH were arranged in a reticular network, short strands and loose fascicles longer than those in classical cases, hence bearing a great resemblance to a soft tissue myoepithelioma (figure 2A,B). Furthermore, in one AFH with sclerosing matrix (case 10), nuclear palisading arrangement was focally seen and accompanied by prominent perivascular hyalinisation with scattered deposition of haemosiderin pigments (figure 2C,D), reminiscent of an ancient schwannoma. Despite no nuclear atypia in four cases, striking nuclear pleomorphism was focally seen in the aggregates of bizarre hyperchromatic tumour cells in two cases (Figure 3A). Moderate nuclear pleomorphism was present in seven cases (figure 1B), including one (case 6) focally concomitant with Ewing sarcoma-like small cell histology (figure 3B). Mitoses varied considerably from 0 to 66 (median 2, mean 8.3) per 10 HPFs. There were osteoclast-like giant cells in two cases, which were easily identified in the pseudoangiomatoid spaces, probably reflective of their reactive nature (figure 3C).
Immunohistochemically, 8 of 10 AFH cases were positive for desmin, 6 of 10 cases for EMA and 9 of 9 for CD99, including the myoepithelioma-like case with a variable extent of reactivity for these three markers.
EWSR1-CREB1 fusion transcript was detected by RT-PCR in nine cases, including the schwannoma-like AFH, and a type II EWSR1-ATF1 fusion was only identified in a myoepithelioma-like AFH (figure 4A). The products of EWSR1-CREB1 in selected cases and EWSR1-ATF1 fusion were confirmed by direct sequencing (figure 4B,C). To ascertain the rearranged EWSR1 gene, both schwannoma-like and myoepithelioma-like AFHs were also cross-validated by break-apart FISH (figure 4D), which also confirmed another AFH (case 12) failed in the RT-PCR assay. However, two cases excised more than 10 years ago (cases 11 and 13) were non-informative by RT-PCR and FISH to detect rearranged EWSR1 or FUS. As the negative controls, two aneurysmal fibrous histiocytomas were neither positive for EWSR1 or FUS rearrangement by FISH nor for EWSR1-AFT1 or EWSR1-CREB1 gene fusion by RT-PCR assay.
As an intermediate mesenchymal malignancy, AFH usually behaves indolently with approximately 2%–10% local recurrence and <1% metastasis.1 ,2 The long-term prognosis of AFH does not correlate well with histomorphological variables,1 ,2 although nine cases in this study did not develop relapsed disease after a range of follow-up up to 108 months. The anatomical distribution of our cases with uncommon clinicopathological features was consistent with previously reported experiences on ordinary AFHs, being most frequent in the subcutis of extremities, followed closely by trunk and head and neck.1 ,2 However, thanks to the identification of gene fusions, rare AFHs originating in a variety of non-somatic locations were increasingly reported at an accelerated pace, such as the lung,9 ,10 mediastinum,11 ,12 ovary,4 brain13 ,14 and bone15. The vast majority of AFHs occur in the first two decades of life,2 ,3 while four cases (30.8%) in this series were diagnosed in patients aged over 20 years. Interestingly, the AFHs in these adults unusually displayed extensive sclerosis in three cases and prominent myxoid changes with reticular architecture in one case, hence posing diagnostic challenges if molecular testing had not been performed. However, it is premature to tell whether these stromal alterations were time-dependent although older patients were more likely to have long-standing diseases.
In our 11 AFHs with molecular confirmation, gene rearrangement only involved EWSR1, rather than FUS. When in doubt, the fusion partner genes need to be identified in these challenging cases, given the growing number of tumour types known to harbour rearranged EWSR1 gene.16 ,17 By using RT-PCR, EWSR1-CREB1 represented the commonest fusion type (90%), and EWSR1-ATF1 was only detected in one myoepithelioma-like AFH. The frequencies of different fusions in variant cases were similar to prior reports on ordinary AFHs,3 ,5 ,18 suggesting no obvious impact of fusion types on the histomorphological features, although Chen et al4 observed a tendency of EWSR1-ATF1 towards non-somatic origins and myxoid stroma. However, it is advised that molecular testing be interpreted in a clinicopathological context before the ascertainment of AFH because the same EWSR1-CREB1 and/or EWSR1-ATF1 fusions may involve other tumour entities with entirely different histology and immunophenotypes.19 For instance, soft tissue and gastrointestinal clear cell sarcomas are well known to harbour such gene fusions, while, unlike AFHs, they exhibit diffuse S100 reactivity, more or less melanocytic differentiation, and importantly, aggressive behaviour.19–21 Actually, it has become explicit that identical chimeric gene products can exist as hallmark molecular aberrations among tumour types of different lineages, such as ETV6-NTRK3 in infantile fibrosarcomas,22 mammary secretory carcinomas or analogues in the salivary glands23 ,24 and a minor subset of leukaemia.25 ,26
Including four solid variants lacking pseudoangiomatous spaces, most AFHs possessed both lymphoplasmacytic cuffs and fibrous pseudocapsules, serving useful clues to call for molecular testing in histologically perplexing cases. Notably, peculiar reticular and palisading arrangements of tumour cells were observed in one case each with prominent myxoid and sclerosing stroma, respectively, thereby imparting a myoepithelioma-like or schwannoma-like appearance. Although schwannomas can be easily differentiated from AFHs by negativity for S100 without molecular testing, combinatorial RT-PCR and FISH assays uniquely documented EWSR1-ATF1 fusion in the myoepithelioma-like AFH. This case was reminiscent of a myxoid and reticular AFH of the mediastinum with the more common EWSR1-CREB1.12 More intriguingly, one soft tissue myoepithelioma27 and a handful of primary pulmonary myxoid sarcomas28 with myxoid and reticular histology were recently shown to harbour EWSR1-ATF1 and EWSR1-CREB1, respectively. However, we are confident of the fidelity of our myoepithelioma-like case as a variant AFH, given its minor focus of syncytial growth without expression of S100, GFAP, p63 and cytokeratin (data not shown). Additionally, striking nuclear pleomorphism (cases 1 and 4) and small round cells (case 6) might disguise these AFHs as pleomorphic or Ewing sarcomas.3 ,18 The latter is particularly prone to be misled in the presence of CD99 reactivity, extraordinarily increased mitoses and FISH-documented rearranged EWSR1 alone.3
In summary, AFHs with prominent histomorphological alterations are not common and may pose challenges to distinguish from other histological mimics with different clinical behaviour. Definite diagnosis of these variant AFHs can be achieved using complementary RT-PCR and FISH assays in an appropriate clinicopathological context.
Take home messages
Most angiomatoid fibrous histiocytomas (AFH) preserve fibrous pseudocapsules and lymphoplasmacytic cuffs as useful clues to call for molecular confirmation in challenging cases.
AFHs rarely display prominent histological alterations in the stromal matrix, growth pattern and cytomorphology, hence mimicking myoepitheliomas, schwannomas, pleomorphic sarcomas or Ewing sarcomas.
Combinatorial RT-PCR and fluorescence in situ hybridisation assays are diagnostically powerful in variant AFHs by documenting EWSR1-CREEB1, EWSR1-ATF1 or FUS-ATF1 fusion when interpreted in a clinicopathological context.
The authors are grateful to the Chang Gung Genomic Core Laboratory for technical assistance (CMRPG880251).
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online supplement
This work has been presented in part in the 102nd annual meeting of USA and Canadian Academy of Pathology, Baltimore, USA, March 2–8, 2013.
Contributors Y-CK and H-YH conceived and designed the study. S-CY and H-YH developed methodology. Y-CK, JL, H-CT, C-FL, J-WT and F-MF acquired data. Y-CK, JL, K-WL and H-YH analysed and interpreted data. Y-CK and H-YH write and revised the manuscript .H-YH supervised the study. All authors read and approved the final manuscript.
Funding This work was supported in part by Chang Gung Hospital (CMRPG8A0321,2 to H-YH) and Wan Fang Hospital, Taipei Medical University (101swf04 to Y-CK) of Taiwan.
Competing interests None.
Ethics approval Institutional Review Boards of Taipei Medical University (201208040) and Chang Gung Memorial Hospital (101-3392B).
Provenance and peer review Not commissioned; externally peer reviewed.