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Review

Wilms tumor 1 (WT1) protein: Diagnostic utility in pediatric tumors Lucia Salvatorelli a,∗ , Rosalba Parenti b , Giorgia Leone c , Giuseppe Musumeci d , Enrico Vasquez a , Gaetano Magro a a Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia, Azienda Ospedaliero-Universitaria “Policlinico-Vittorio Emanuele”, Anatomic Pathology Section, University of Catania, Catania, Italy b Department of Biomedical and Biotechnological Sciences, Physiology Section, University of Catania, Catania, Italy c Anatomic Pathology, Hospital of Sondrio, Sondrio, Italy d Department of Biomedical and Biotechnological Sciences, Human Anatomy and Histology Section, School of Medicine, University of Catania, Catania, Italy

a r t i c l e

i n f o

Article history: Received 18 December 2014 Received in revised form 15 March 2015 Accepted 20 March 2015 Available online xxx Keywords: WT1 Tumors Children Adolescents Review

a b s t r a c t Despite Wilms tumor 1 (WT1) protein was originally considered as a specific immunomarker of Wilms tumor, with the increasing use of immunohistochemistry, there is evidence that other tumors may share WT1 protein expression. This review focuses on the immunohistochemical profile of WT1 protein in the most common malignant tumors of children and adolescents. The variable expression and distribution patterns (nuclear vs cytoplasmic) in the different tumors, dependent on the antibodies used (anti-C or N-terminus WT1 protein), will be emphasized by providing explicative illustrations. Potential diagnostic pitfalls from unexpected WT1 protein expression in some tumors will be discussed in order to avoid diagnostic errors, especially when dealing with small biopsies. © 2015 Elsevier GmbH. All rights reserved.

Introduction The Wilms tumor 1 (WT1) gene on chromosome 11p13, encodes a zinc-finger transcription factor with developmental, tumor suppressor and oncogenic properties (Call et al., 1990; Gessler et al., 1990; Hohenstein and Hastie, 2006; Lee and Haber, 2001; Parenti et al., 2014b). These different, occasionally opposite, functions may be explained if we consider that various isoforms of WT1 do exist, as a result of alternative splicing (Menke et al., 1998; Lee and Haber, 2001; Hohenstein and Hastie, 2006; Roberts, 2006; Hartkamp and Roberts, 2008; Huff, 2011). Several studies have shown that WT1 plays an important role during human development, being expressed in several tissues, both at nuclear and cytoplasmic level. In this regard, nuclear immunohistochemical expression is frequently found in metanephric and mesonephric glomeruli, primary sex cords, gonadic stroma, mesothelial and submesothelial cells, while cytoplasmic expression is usually seen in developing skeletal muscles, myocardium, radial glia of spinal cord and cerebral cortex, sympathetic neuroblasts, adrenal cortical cells and endothelial cells of blood vessels (Pritchard-Jones et al., 1990; Sharma et al., 1992; Armstrong et al., 1993; Mundlos et al., 1993;

∗ Corresponding author. E-mail address: [email protected] (L. Salvatorelli).

Ramani and Cowell, 1996; Charles et al., 1997; Parenti et al., 2013, 2014a, 2015). With regard to neoplastic tissues, WT1 protein has been found in several tumors with variable immunostaining patterns, i.e., exclusively nuclear, cytoplasmic or both, according to the antibodies used (anti-C or N-terminus WT1 protein) (Carpentieri et al., 2002; Nakatsuka et al., 2006; Schittenhelm et al., 2010; Bisceglia et al., 2011a,b; Salvatorelli et al., 2011; Singh et al., 2012; Magro et al., 2014a,b, 2015c,d). The different sub-cellular distribution of WT1 protein is consistent with its nucleo-cytoplasmic shuttling properties (Niksic et al., 2004), suggesting its involvement as regulator of transcription or post-transcriptional processes, depending on the tumor tissue and environmental contex (Hohenstein and Hastie, 2006). Interestingly, in some tumors, such as Wilms tumor, ovarian, mesothelial neoplasms, Sertoli cell tumor, and rhabdomyosarcoma, the variable nuclear and cytoplasmic WT1 staining may be explained by assuming that the expression of this transcription factor in some neoplastic tissues mirrors its normal developmental regulation (Parenti et al., 2013, 2014a; Magro et al., 2015c). Recently, we have also shown that WT1 shows an oncofetal expression pattern, being abundantly detected in developing and neoplastic skeletal muscle tissues, while its expression is downregulated in adult normal skeletal muscle tissues (Salvatorelli et al., 2011; Magro et al., 2015c). However, over the last years there is increasing evidence showing that WT1 protein is expressed “de

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novo” in some tumors (colorectal, breast cancer, and brain tumors) which arise from tissues that normally do not, suggesting a potential oncogenic role in these neoplasms (Loeb et al., 2001; Oji et al., 2002, 2004a,b; Koesters et al., 2004; Hohenstein and Hastie, 2006; Kang et al., 2010; Oue et al., 2011). The diagnosis of benign and malignant pediatric tumors is often challenging, mainly due to the fact that some of these tumors share overlapping morphological and/or immunohistochemical features, with the possibility of misdiagnosing a benign lesion with a malignant tumor or vice versa (Magro et al., 2005, 2008, 2011; Alaggio et al., 2009). In addition a subset of malignant tumors may present unusual clinical, morphological and immunohistochemical features which make difficult their recognition, especially when dealing with small biopsies. Therefore there is an increasing need of highly sensitive and specific immunomarkers which can be exploitable in daily practice in order to achieve a correct diagnosis (Magro et al., 2015a,b). Among these immunomarkers, WT1 protein, traditionally considered as specific for Wilms tumor, is expressed in a wide variety of other neoplasms of children and adolescents, even if with a variable cellular distribution (nuclear vs cytoplasmic expression). The aim of this review is to focus on the immunohistochemical expression of WT1 protein in the most common tumors of children and adolescents. The potential diagnostic role of WT1 protein will be emphasized, providing illustrations and discussion about its usefulness in the differential diagnosis. Potential diagnostic pitfalls, which can arise from unexpected WT1 protein immunoreactivity, will be also discussed. WT1 antibodies: Nuclear versus cytoplasmic immunoreactivity For a long time, it was believed that immunohistochemical expression of WT1 protein was exclusively limited to the nucleus and that the cytoplasmic localization was an artefact, likely due

to cross-reactivity of the antibodies used. Nuclear staining has been obtained especially with antibodies directed against the Cterminal portion of WT1 protein (clone WT 1C19) (Ramani and Cowell, 1996; Charles et al., 1997). However with the advent of new available antibodies against the N-terminal portion of WT1 protein (clone WT 6F-H2), more recent studies have shown that cytoplasmic WT1 staining can be obtained in the nucleus or cytoplasm, or concurrently in both nucleus and cytoplasm (Carpentieri et al., 2002; Bisceglia et al., 2011a; Parenti et al., 2013, 2014a; Magro et al., 2014a,b, 2015c,d). This variable immunoreactivity may be explained assuming that WT1 acts as regulator of either transcriptional or translational processes, shuttling between the nucleus and the cytoplasm (Niksic et al., 2004; Hohenstein and Hastie, 2006). WT1 protein expression in tumors of children and adolescents Wilms tumor (nephroblastoma) It is a malignant pediatric embryonal neoplasm that occurs as a result of a disturbance of cellular differentiation of the metanephric blastema (Davies et al., 2004; Chau and Hastie, 2012; Miller-Hodges and Hohenstein, 2012; Al-Hussain et al., 2014). Accordingly, this neoplasm replicates, at least partially, the morphology of the developing metanephros (Fig. 1A and B). Wilms tumor is the most common genitourinary tumor of childhood, with most cases diagnosed in children between 2 and 4 years of age. Both kidneys are equally affected, and there is the possibility of synchronous or metachronous bilateral involvement in 5–10% of cases. Classically, Wilms tumors presents as an abdominal mass, with abdominal pain, hematuria and hypertension. Histologically, the majority of Wilms tumors usually exhibits triphasic histological components, consisting of blastematous, epithelial, and stromal components (Al-Hussain et al., 2014) (Fig. 1C). The blastematous component

Fig. 1. (A and B) Metanephros of human fetus of 11 weeks of gestational age. (A) Histological illustration showing developing metanephros. The sub-capsular nephrogenic zone, containing blastema and developing epithelial structures, is seen (hematoxylin and eosin, original magnification ×80); (B) Nuclear WT1 expression is found in the blastema and podocytes of developing glomeruli (immunoperoxidase method with WT1 C-terminus antibodies, original magnification ×80). (C and D) Wilms tumor. (C) Histological examination showing the triphasic components: blastema (B), epithelial structures and stromal component (S) (hematoxylin and eosin, original magnification ×100); (D) WT1 is expressed in the nuclei of the blastematous component, in the cytoplasm of stromal cells, while it is lacking in the epithelial structures (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×100).

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Fig. 2. Intra-abdominal desmoplastic small round cell tumor of a 11 year-old female. (A) Tumor nests surrounded by fibro-sclerotic stroma are typical features (hematoxylin and eosin, original magnification×80); neoplastic cells showing cytoplasmic desmin (B) and cytokeratin (C) expression (B and C: immunoperoxidase method, original magnification×150); (D) Nuclear WT1 staining is obtained with WT1 C-terminus antibodies (immunoperoxidase method, original magnification ×200).

consists of densely packed small round blue cells with scanty cytoplasm, dark nuclei and frequent mitotic figures. They are usually arranged in several growth patterns, including diffuse, nodular and cord-like patterns. Notably, some cases of Wilms tumors are composed almost exclusively of the above mentioned blastematous component and, thus, may result diagnostically challenging, especially when dealing with small biopsies (differential diagnosis with other small round blue cell tumors). The epithelial component is often composed of tubules, papillary and glomerular-like structures which are closely reminiscent of normal nephrogenesis. Primitive rosette-like structures, representing early tubular forms and cysts lined by primitive columnar or cuboidal cells can be usually seen. The stromal component exhibits a variety of patterns of differentiation. Most tumors are composed of spindle-shaped cells set in a myxoid stroma, vaguely resembling embryonal mesenchyme. Heterologous stromal elements, such as skeletal muscle, cartilage, bone, fat, or more rarely neuroglia and mature ganglion cells can be seen. Skeletal muscle in various stages of differentiation, including rhabdomyoblasts, represents the most common divergent line of differentiation. Identification of anaplastic cells (anaplasia), i.e. large neoplastic cells with nuclear pleomorphism and multipolar mitotic figures, is important because it may have prognostic implications (favorable vs unfavorable histology), according to its tumor extension (focal vs diffuse). Anaplasia is usually seen in about 5% of Wilms tumors and seems to be associated with a poor prognosis and increased risk of treatment failure, especially when it is diffusely distributed. The immunohistochemical profile depends on the different tumor components examined. Blastematous component shows diffuse expression of vimentin and WT1, while a variable staining is found with CD56, CD57, cytokeratins, EMA, desmin and PAX2 (Ramani and Cowell, 1996; Charles et al., 1997; Nakatsuka et al., 2006; Vasei et al., 2008; Davis et al., 2011; Arnold et al., 2014; Sehic et al., 2014). The epithelial elements stain for cytokeratin,

EMA, CD56 and variably for PAX2 and WT1 (Ramani and Cowell, 1996; Charles et al., 1997; Vasei et al., 2008; Davis et al., 2011). The cells of the stromal component are usually vimentin positive, while the heterologous skeletal muscle component is reactive for desmin, myogenin and MyoD1. Among the above mentioned markers, WT1 is certainly the most sensitive and specific marker for the diagnosis of Wilms tumor, being dected in more than 90% of cases (Ramani and Cowell, 1996; Charles et al., 1997; Nakatsuka et al., 2006). WT1 protein is expressed mainly at nuclear level, using antibodies directed both to C-terminal or N-terminal portions of the WT1 protein (Ramani and Cowell, 1996; Charles et al., 1997; Nakatsuka et al., 2006), with an immunoreactivity which can be detected in blastematous, and less frequently, in epithelial and stromal cells. This immunoreactivity mirrors the developmental expression of WT1 protein during human nephrogenesis (Fig. 1B). A concurrent or exclusively (more often in blastematous and stromal cells) cytoplasmic WT1 expression can be found, especially by using WT1 N-terminus antibodies (Nakatsuka et al., 2006) (Fig. 1D). Similarly for desmoplastic small round cell tumor, only nuclear staining should be considered as specific for diagnosis of Wilms tumor. Desmoplastic small round cell tumor This highly aggressive tumor, originally described as multiple intra-abdominal tumor masses, is now recognized to arise from many other sites, including pleura, kidney, ovary, scrotum, meninges, bone, scalp, paranasal sinuses, pancreas, and parotid gland (Antonescu and Ladanyi, 2013). Because of its predominant location in mesothelium-lined cavities and its immunohistochemical profile, a primitive origin from mesothelial/sub-mesothelial cells was originally postulated, but later denied. This tumor usually occurs in children and adolescents, with male predilection. Abdominal pain and multiple abdominal masses, without a primary organ involved, are the usual presenting findings (Antonescu and

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Fig. 3. Renal rhabdoid tumor of 1 year-old female (A) histological examination showing the typical rhabdoid cells with abundant eosinophilic cytoplam (hematoxylin and eosin, original magnification ×80); neoplastic cells are positive for cytokeratin (B), while they typically lack nuclear INI-1 expression (C); intratumoral lymphocytes are stained and served as internal control (immunoperoxidase method, original magnification ×150); (D) Unusual nuclear WT1 staining is obtained with WT1 N-terminus antibodies; endothelial cells of intra-tumoral vessels show cytoplasmic staining, and served as internal control (immunoperoxidase method, original magnification ×200).

Ladanyi, 2013). The most common sites of origin are the mesentery, omentum, surface of the liver, and pelvic peritoneum (Antonescu and Ladanyi, 2013). Histologically, it is composed of variably-sized nests, trabeculae, or lobules of malignant small cells, usually separated by a prominent fibro-sclerotic stroma (Fig. 2A). Neoplastic cells are round in shape, with scant cytoplasm, indistinct cell borders and hyperchromatic round to oval, or slightly angulated, nuclei that have finely granular chromatin and small nucleoli. Central necrosis and calcification may be seen within the nests (Fig. 2A). Mitoses are usually observed. In some cases, unusual morphological features, such as rhabdoid or signet ring appearance, as well as glandular or pseudorosettes formation have been described (Antonescu and Ladanyi, 2013). Immunohistochemically, desmoplastic small round cell tumor is characterized by a polyphenotypic profile, with co-expression of vimentin, desmin (Fig. 2B), epithelial markers (cytokeratin; epithelial membrane antigen) (Fig. 2C), and WT1 (Fig. 2D) (Antonescu and Ladanyi, 2013). Although other markers, such as CD99, smooth muscle actin, neuron-specific enolase, CD57 and synaptophysin are variably found, the co-expression of epithelial markers, desmin and WT1 have a major diagnostic value. With regard to WT1, it should be emphasized that most cases (>90%) of desmoplastic small round cell tumor show strong nuclear staining with antibodies directed against the C-terminal portion of WT1 protein (clone WT1 C19) (Barnoud et al., 2000; Hill et al., 2000). This unexpected WT1 positivity was originally considered as an evidence of the possible origin of the tumor from mesothelial cells, which are WT1-positive since fetal development (Parenti et al., 2013, 2015). However, the lack of staining for other mesothelial markers, such as cytokeratins 5/6 and calretinin, argues against this hypothesis. It is widely accepted that the aberrant nuclear expression of WT1 protein in this tumor is due to a recurrent chromosomal translocation t(11;22)(p13;q12),

which can be found in about 90% of cases (Antonescu and Ladanyi, 2013). Two genes, EWSR1 gene and WT-1 gene, are fusion partners, resulting in EWSR1-WT1 fusion transcript (Antonescu and Ladanyi, 2013). Some authors state that only nuclear staining obtained with the WT1 C-terminus antibodies (clone WT C19) is of diagnostic utility in diagnosis of desmoplastic small round cell tumor, because they are predictive of the EWS-WT1 translocation with high sensitivity and specificity (Hill et al., 2000; Carpentieri et al., 2002). Infact nuclear staining is found with the former antibodies in >95% of cases, in contrast to a minority of cases which results to be positive also with WT1 N-terminus antibodies (Carpentieri et al., 2002; Nakatsuka et al., 2006; Murphy et al., 2008). It has been suggested that the unusual nuclear WT1 immunoreactivity with N-terminus antibodies is likely due to novel fusion transcripts (Murphy et al., 2008). Interestingly, apart from nuclear staining, a weak to moderate cytoplasmic WT1 positivity has been reported in some cases of desmoplastic small round cell tumor, especially by using N-terminus antibodies (Nakatsuka et al., 2006; Wang et al., 2007; Bisceglia et al., 2011a). Based on these findings, we suggest the use of WT1 C-terminus (clone WT C19) antibodies for the diagnosis of desmoplastic small round cell tumor, and that only a strong nuclear staining should be considered as a positive result. Malignant rhabdoid tumor Malignant rhabdoid tumor is a highly aggressive neoplasm that usually occurs in the kidney of children, but less frequently may arise from other sites, including central nervous system, somatic soft tissues, abdomen, pelvis, retroperitoneum, liver, heart, and gastrointestinal tract (Alaggio et al., 2012). Interestingly, renal and nervous system tumors occur frequently in children younger than

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Fig. 4. (A and B) Embryonal rhabdomyosarcoma, botryoid type, of the external auditory canal of a 5 year-old boy. (A) Histological examination showing a proliferation of small round to spindle cells set in a myxoid stroma, beneath the squamous epithelium (hematoxylin and eosin, original magnification ×60). (B) Neoplastic cells showing strong and diffuse cytoplasmic WT1 staining (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×60). (C and D) Spindle cell/sclerosing rhabdomyosarcoma of the oral cavity. (C) Histological examination showing a proliferation of spindle cells set in fibrous stroma (hematoxylin and eosin, original magnification ×60). (D) Neoplastic cells showing strong and diffuse cytoplasmic WT1 staining (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×60).

10 years, while soft tissue tumors are usually diagnosed in fetuses, newborns and young children (Alaggio et al., 2012). A variable number of cases (15–85%), including congenital cases, are metastastic at presentation, and pursue a fatal course (Alaggio et al., 2012). Histologically, it is composed of round/epithelioid to polygonal cells, variably arranged in solid or trabecular growth patterns. Characteristically, neoplastic cells have abundant, deeply eosinophilic cytoplasm with a paranuclear eosinophilic, PAS-positive inclusion and large, round, vescicular nuclei with finely dispersed chromatin containing a prominent eosinophilic nucleolus (Fig. 3A). Mitoses and necrosis are commonly seen. A minor tumor component is represented by smaller, round, undifferentiated cells with scant cytoplasmic rim. In some cases, this unusual cellular component may be prominent, posing serious differential diagnostic problems with small round cell tumors, especially Ewing’s sarcoma/peripheral primitive neuroectodermal tumors (Sigauke et al., 2006; Alaggio et al., 2009; Machado et al., 2010). Immunohistochemically, malignant rhabdoid tumor, like desmoplastic small round cell tumor, exhibits a poliphenotypic profile, with variable co-expression of different markers, including vimentin, cytokeratin (Fig. 3B), epithelial membrane antigen, and CD99 (Alaggio et al., 2012). However the most useful diagnostic marker is the complete absence of nuclear immunoreactivity for INI1 protein (Hoot et al., 2004; Sigauke et al., 2006; Judkins, 2007; Machado et al., 2010) (Fig. 3C). Additional markers, such as muscle specific actin, alpha-smooth muscle actin, S100 protein, synapthophisin, CD56 and also WT1 can be occasionally expressed (Ramani and Cowell, 1996; Charles et al., 1997; Alaggio et al., 2012). As far as WT1 protein expression is concerned, immunostaining, exclusively nuclear or nucleo-cytoplasmic, can be detected using antibodies directed against the C-terminal portion of WT1 protein (clone WT C19) also in some cases of malignant rhabdoid tumor (Ramani and Cowell, 1996; Charles et al., 1997). We have experience of similar results also by using antibodies against the N-terminal portion (clone WT 6F-H2) (Fig. 3D).

Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a malignant tumor composed of cells which show variable morphological, immunohistochemical and ultrastructural evidence of skeletal muscle differentiation. Based on morphological, immunohistochemical and molecular features, at least four major subtypes can be recognized: (i) embryonal; (ii) alveolar; (iii) spindle cell/sclerosing; (iv) pleomorphic. While pleomorphic rhabdomyosarcoma is typically a tumor of adults, the other subtypes occur predominantly in children and adolescents. Embryonal rhabdomyosarcoma, the most common subtype, occurring in children aged 50% of neoplastic cells) of spindle-shaped cells arranged in whorls or in a fascicular/

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Fig. 5. (A) Poorly differentiated neuroblastoma: neoplastic cells lacking WT1 staining; intra-tumoral endothelial cells of blood vessels show cytoplasmic staining, and served as internal control (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×80); (B) Intermixed ganglioneuroblastoma: immature ganglion cells and Schwann cells of the stromal component, show cytoplasmic WT1 staining (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×100).

storiform growth pattern closely reminiscent of fibrosarcoma or leiomyosarcoma (Parham et al., 2012) (Fig. 4C). A variable amount of extracellular sclerosis is seen and, when extensive, the term “sclerosing rhabdomyosarcoma” can be used. Alveolar rhabdomyosarcoma is a distinct subtype of rhabdomyosarcoma usually associated with aggressive behavior. It affects adolescents and young adults, with a peak incidence at 10–25 years of age. The most common sites of occurrence are the deep soft tissues of the extremities and axial musculature. Histologically, it is characterized by cellular nests separated by fibro-vascular septa. Neoplastic cells, with scant cytoplasm and large hyperchromatic nuclei, are mainly discohesive in the center of the nests, while they are attached to the fibro-vascular septa at the periphery of the nests. This histological growth pattern is designated with the term “alveolar” because it is vaguely reminiscent of lung alveoli. Some cases, which lack the alveolar growth pattern, are classified as “solid variant”, being predominantly composed of sheets of closely packed neoplastic cells with no intervening fibrous septa. Interestingly, some cases of rhabdmyosarcoma may have overlapping or mixed morphology, i.e. areas of embryonal and alveolar subtypes may coexist in the same tumor. Immunohistochemically, virtually all cases of rhabdomyosarcoma, whatever the subtype, are positive, albeit with a variable extension, to desmin, myogenin and MyoD1, currently considered the most reliable markers of this tumor (Parham et al., 2012). Although it is true that immunohistochemistry staining pattern is not reliable in subtyping rhabdomyosarcoma, there is evidence that alveolar rhabdomyosarcoma usually exhibits a more diffuse staining for both desmin and myogenin compared with embryonal rhabdomyosarcoma. Among these markers, myogenin and MyoD1 are considered highly specific markers of skeletal muscle differentiation, as desmin can be expressed by several myofibroblastic/leiomyomatous lesions (Parham et al., 2012). Interestingly, both embryonal and alveolar rhabdomyosarcoma may also variably express CD99, cytokeratin, S100 protein, alpha-smooth muscle actin, and neuroendocrine markers, such as chromogranin A and synaptophysin (Parham et al., 2012). In addition some cases of rhabdomyosarcoma have been reported to be WT1-positive. In this regard it should be emphasized that nuclear WT1 staining

can be focally demonstrated only in a few cases, by using WT1 Cterminus antibodies (Barnoud et al., 2000). Conversely, with the advent of new available WT1 N-terminus antibodies (clone WT 6F-H2), several studies have reported a diffuse and strong cytoplasmic expression in embryonal (Fig. 4B), sclerosing/spindle cell (Fig. 4D) and alveolar subtypes of rhabdomyosarcoma (Carpentieri et al., 2002; Sebire et al., 2005; Bisceglia et al., 2011a,b; Magro et al., 2015c). This unexpected finding is explained by the detection of WT1 in the cytoplasm of human developing myoblasts and myotubes during the early phases of skeletal myogenesis (Salvatorelli et al., 2011; Parenti et al., 2013, 2015; Magro et al., 2015c,d; Musumeci et al., 2015), suggesting its potential role in the molecular mechanisms that regulate skeletal muscle differentiation. Although we admit that the diagnosis of rhabdomyosarcoma is based on the demonstration of myogenic markers (desmin, myogenin, MyoD1), the strong and diffuse cytoplasmic WT1 staining can be useful in confirming the diagnosis when dealing with a small round cell tumor morphologically consistent with a rhabdomyosarcoma (i.e., identification, even if only focally, of cells with the features of rhabdomyoblasts) but which lacks myogenin and MyoD1 (Parham et al., 2012). In our experience, a strong and diffuse cytoplasmic WT1 expression (WT1 N-terminus-clone WT 6F-H2) can be exploitable as an adjunct helpful immunomarker of rhabdomyosarcoma versus other small round cell tumors (Magro et al., 2015c,d). Neuroblastic tumors Neuroblastic tumors occur in children and adolescents, especially from the adrenal gland, retroperitoneum or, more rarely, in the posterior mediastinum. They represent a heterogeneous group of lesions characterized by a wide morphological spectrum which reflects a different degree of differentiation from immature neuroblastic cells to mature ganglionic cells (Rosai, 2011). According to the degree of maturation, the following histological categories are defined: (i) neuroblastoma (Schwannian stroma-poor tumors), including undifferentiated, poorly differentiated and differentiating neuroblastomas; (ii) ganglioneuroblastoma (Schwannian stroma-rich tumors), including intermixed and nodular

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Fig. 6. Infantile fibrosarcoma of the leg of a 4 month-old male infant. (A) Histological examination showing spindle cells arranged in intersecting fascicles (hematoxylin and eosin, original magnification ×80); (B) neoplastic cells showing strong and diffuse cytoplasmic WT1 staining (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×100).

ganglioneuroblastomas; (iii) ganglioneuroma (Schwannian stroma predominant tumors), including maturing and mature ganglioneuromas (Rosai, 2011). Neuroblastoma consists of small, round cells, with scant cytoplasm and round, hyperchromatic nuclei. The tumor cells sometimes are arranged around a fibrillar area (neuropil), resulting in the so-called Homer Wright rosettes. If ≤5% or more than 5% of the tumor cells shows ganglionic differentiation, the neuroblastoma is classified, respectively, as poorly differentiated or differentiating neuroblastoma; in contrast when neoplastic cell do not show any differentiation toward ganglionic cells and the neuropil is almost or completely absent, the neuroblastoma is classified as undifferentiated neuroblastoma (Rosai, 2011). Ganglioneuroblastoma exhibits features of differentiation that are intermediate between neuroblastoma and ganglioneuroma. Histologically, two distinct entities can be recognized, intermixed and nodular. In both variants there is a ganglioneuromatous component, but in the former collection of immature ganglion cells are interspersed among the Schwannian stroma, while in the latter, in addition to the ganglioneuromatous component, there is an area of neuroblastoma (Rosai, 2011). Ganglioneuroma rapresents the fully differentiated member of neuroblastic tumors, being composed of mature or maturing ganglion cells surrounded by fascicles of Schwann cells (Rosai, 2011). Several morphological, immunohistochemical and in vitro studies support the concept that childhood peripheral neuroblastic tumors recapitulate the developmental stages of normal peripheral sympathetic nervous system (Magro et al., 1995, 2000; Magro and Grasso, 1997, 2001; De Preter et al., 2007; Hoehner et al., 1998). Neuroblastoma is traditionally considered as a WT1-negative tumor, with only a few studies reporting focal and weak nuclear WT1 staining in neuroblastoma (Barnoud et al., 2000; Sebire et al., 2005; Carpentieri et al., 2002). A more recent study showed WT1 expression preferentially in ganglioneuroblastoma

and ganglioneuroma compared with neuroblastoma (Wang et al., 2007). In our experience, by using antibodies against the Nterminal portion of WT1 protein (clone WT 6F-H2), the more undifferentiated neuroblastic component in both neuroblastoma and ganglioneuroblastoma is usually WT1-negative (Fig. 5A), while a variable cytoplasmic WT1 staining is found in ganglion cell component of both ganglioneuroblastoma and ganglioneuroma (Fig. 5B). Congenital/infantile fibrosarcoma It is a malignant tumor currently classified in the category of the intermediate (rarely metastasizing) neoplasms, which typically occurs in the first 2 years of life, with a significant number of cases diagnosed at birth or antenatally (Coffin and Alaggio, 2012). The most commonly involved sites are soft tissues of the trunk and distal extremities, with only rare cases reported in the retroperitoneum. Prognosis is favorable, with distant metastases documented in only a few cases (Coffin and Alaggio, 2012). Histologically, it is a highly cellular neoplasm composed predominantly of spindle cells variably arranged in fascicles, focally exhibiting a herringbone pattern (Fig. 6A). Areas with more immature round to polygonal cells can be seen. Generally, neoplastic cells show only a mild to focally moderate degree of nuclear atypia. Mitoses are usually numerous, even if atypical mitoses are absent. A hemangiopericytoma-like vascular pattern and hypocellular fibrotic areas can be seen (Coffin and Alaggio, 2012). Although congenital/infantile fibrosarcoma may variably express, more frequently with focal extension, alpha-smooth muscle actin and/or desmin, highly specific markers are not available for this tumor. Accordingly, the diagnosis is frequently of exclusion, being mainly based on negative results for specific lineage markers such as desmin, myogenin, CD34, S-100, HMB-45, and

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Fig. 7. (A and B) Blastemal-dominant Wilms tumor. (A) A close resemblance with desmoplastic small round cell tumor is evident (hematoxylin and eosin, original magnification ×60). (B) Blastematous cells showing strong and diffuse nuclear WT1 staining (immunoperoxidase method with WT1 C-terminus antibodies, original magnification ×100). (C and D) Desmoplastic small round cell tumor. (C) Typical histological picture of desmoplastic small round cell tumor (hematoxylin and eosin, original magnification ×60). (D) Neoplastic cells lacking WT1 expression (immunoperoxidase method with WT1 N-terminus antibodies, original magnification ×100).

cytokeratins (Coffin and Alaggio, 2012). Given the non-specificity of immunohistochemistry, the diagnosis of congenital/infantile fibrosarcoma is usually confirmed, especially for morphologically and/or immunohistochemically ambiguous cases, by the identification of the recurrent translocation t(12;15) (p13;q25) with an ETV6-NTRK3 gene fusion (Bourgeois et al., 2000; Sheng et al., 2001). Recently, we first showed that congenital/infantile fibrosarcoma is a WT1-positive tumor by using antibodies against the N-terminal portion of WT1 protein (clone WT 6F-H2) (Fig. 6B) (Magro et al., 2014a). In this regard a strong and diffuse cytoplasmic WT1 staining is obtained not only in surgically resected samples but also in small biopsies, suggesting that WT1 can be successfully used in confirming the diagnosis of congenital/infantile fibrosarcoma (Magro et al., 2014a). In this regard, WT1 is useful in daily practice, especially in distinguishing congenital/infantile fibrosarcoma from desmoid-type fibromatosis, a lesion which, albeit may show overlapping morphological and immunohistochemical features with the former, is typically WT1-negative (Magro et al., 2014a). WT1 immunoreactivity: Potential diagnostic pitfalls Although WT1 is a useful marker in the diagnosis of pediatric tumors, an erroneous interpretation of its staining in some clinicopathologic settings may lead pathologists to a misdiagnosis. It is commonly believed that a pediatric small round blue cell tumor with nuclear immunostaining for WT1 protein is a Wilms tumor. However, even if WT1 protein is a highly sensitive immunomarker of Wilms tumor, it is not specific, being expressed in other morphologic mimics. Wilms’ tumor versus desmoplastic small round cell tumor A pediatric renal tumor which expresses WT1 protein in the nuclei of neoplastic cells is considered a Wilms’ tumor. This is

true if an epithelial component can be demonstrated, at least focally, in association with a blastematous and/or stromal component. However differential diagnostic problems may arise when dealing with a blastemal-dominant Wilms tumor, in which the epithelial component is lacking or only focally detectable (Fig. 7A). In this regard, differential diagnostic problems mainly revolves around desmoplastic small round cell tumor which, albeit commonly observed in the mesentery, omentum, surface of the liver, and pelvic peritoneum, may primarily arise in kidney or retroperitoneum of children and adolescents (Wang et al., 2007; da Silva et al., 2009) (Fig. 7C). Moreover there is the possibility that Wilms’ tumor be so large in size to be diagnosed, by radiological imaging, as abdominal or retroperitoneal mass which involves secondarily the kidney. Accordingly, distinguishing a blastemal-dominant Wilms tumor from a desmoplastic small round cell tumor may be challenging (Wang et al., 2007; da Silva et al., 2009; Arnold et al., 2014), especially if pathologist is dealing with a small, round- to ovoid-shaped cell component as the sole cytotype in small biopsies obtained from renal masses. The possibility of misdiagnosing a desmoplastic small round cell tumor with a Wilms tumor is mainly due to the fact both tumors share nuclear WT1 expression by using WT1 (C-terminus; clone WT C19) antibodies. In addition, the confusion may be also enhanced by the evidence that desmin, a marker expressed in >90% of cases of desmoplastic small round cell tumors, is occasionally found in the blastemal component of the Wilms’ tumor, especially with a dot-like, perinuclear staining pattern (Arnold et al., 2014). A correct diagnostic interpretation relies on the differential expression of WT1 protein in the two tumors by using different WT1-antibodes. In this regard, pathologists should keep in mind that the majority (>95%) of desmoplastic small round cell tumors results to be WT1-positive (nuclear staining) only if antibodies against the C-terminal portion (clone WT C19) are used (Fig. 2D) (Hill et al., 2000; Carpentieri et al., 2002; Murphy et al., 2008). In contrast nuclear staining for WT1 is still maintained in

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Fig. 8. Renal rhabdoid tumor of a 1 year-old female. (A–C) Core biopsy. (A) Histological examination showing a proliferation of small, round cells without any feature consistent with rhabdoid tumor. (B) At higher magnification cellular details of neoplastic cells can be better appreciated. (C) Neoplastic cells showing a strong and diffuse nuclear staining for WT1 (A: hematoxylin and eosin, original magnification ×80; B: hematoxylin and eosin, original magnification ×150; C: immunoperoxidase method with WT1 N-terminus antibodies; original magnification ×100). (D) Histological examination of the surgically resected sample, showing that tumor was composed predominantly of the typical rhabdoid cells with abundant eosinophilic cytoplasm, which were lacking in the core biopsy (hematoxylin and eosin, original magnification ×200).

Wilms’ tumor (Fig. 7B), whereas it is lacking in the majority (>97%) of cases of desmoplastic small round cell tumor by using WT1 Nterminus antibodies (clone WT 6F-H2) (Fig. 7D). Based on these considerations, we strongly suggest that when pathologists are dealing with a small round blue cell tumor of the kidney or abdominal/retroperitoneal cavity, WT1 C- and N-terminus antibodies should be included in the immunohistochemical panel, including desmin, cytokeratins and INI1 protein. The last antibodies (antiINI1 protein) are useful in ruling out malignant rhabdoid tumor. If tumor exhibits diffuse and strong nuclear immunoreactivity with WT1 N-terminus antibodies, it is likely a Wilms’ tumor, even if desmin and cytokeratin immunostaining is variably represented and INI1 expression is retained. A similar diagnostic conclusion should be considered if immunostaining is also obtained with WT1 C-terminus antibodies, along with absence of both desmin and cytokeratins and concurrent expression of INI1 protein. Lastly, if tumor exhibits strong nuclear immunoreactivity only with WT1 C-terminus antibodies, in addition to a diffuse staining for desmin, cytokeratins and INI1, the possibility of a desmoplastic small round cell tumor should be seriously considered, especially if the morphological features are suggestive. Molecular tests, especially the detection of EWS-WT1 fusion transcript by reverse transcription polymerase chain reaction (RT-PC), are mandatory in confirming the diagnosis of desmoplastic small round cell tumor in those cases with ambiguous morphology and/or immunohistochemical profile. Wilms’ tumor versus malignant rhabdoid tumor Kidney is one of the most common site of malignant rhabdoid tumor in children (Alaggio et al., 2012). Although this tumor is suspected if the typical cells with rhabdoid morphology are present,

it should be emphasized that a small round cell component may variably be represented throughout the tumor (Fowler et al., 2006; Alaggio et al., 2009). It consists of cells with a lymphocyte-like appearance, showing scant cytoplasm and slightly hyperchromatic, round-shaped nuclei with rare and inconspicuous nucleoli (Alaggio et al., 2009). The identification, at least focally, of larger neoplastic cells with nuclei containing finely dispersed chromatin, and one or two small evident nucleoli, may help in the correct interpretation of tumor histotype (Alaggio et al., 2009). However serious differential diagnostic problems between a blastemal-dominant Wilms tumor and a malignant rhabdoid tumor do exist when dealing with small biopsies from renal tumor masses which contain exclusively/predominantly a small, round cell component (Fig. 8A and B). The potential diagnostic pitfall is mainly due to the fact nuclear WT1 staining can be detected using WT1 Cterminus antibodies in some cases of malignant rhabdoid tumor (Ramani and Cowell, 1996; Charles et al., 1997); in our experience, we have seen a case of renal malignant rhabdoid tumor (Fig. 8D) with a small cell round component at core biopsy, which expressed nuclear staining by using WT1 (N-terminus-clone WT 6F-H2) antibodies (Fig. 8C). As both Wilms’ tumor and rhabdoid tumor may variably stain positively with vimentin and cytokeratins, the most reliable marker helpful in their distinction is INI1 protein. If a renal WT1-positive tumor lacks nuclear immunoreactivity for INI1 protein, the diagnosis of malignant rhabdoid tumor is likely (Hoot et al., 2004; Sigauke et al., 2006; Judkins, 2007; Machado et al., 2010). Based on these considerations, pathologists should be aware that not all WT1-positive renal tumors are Wilms tumors, and when approaching a small biopsy from a renal tumor mass, apart from WT1 (both C- and N-terminus) antibodies, INI protein should be routinely included in the immunohistochemical panel.

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Rhabdomyosarcoma versus other small round cell tumors All subtypes of rhabdomyosarcoma show a diffuse and strong cytoplasmic WT1 staining by using WT1 (N-terminus) antibodies (Fig. 4B and D) (Carpentieri et al., 2002; Sebire et al., 2005; Bisceglia et al., 2011a,b; Magro et al., 2015c,d). A similar staining has also been reported in up 43% of cases with antibodies directed against C-terminus of WT1 protein (Nakatsuka et al., 2006). Although this finding is helpful in confirming diagnosis of rhabdomyosarcoma, especially in tumors with ambiguous immunoprofile, WT1 should not be used alone in daily practice. This is due to the evidence that rhabdomyosarcoma mimics, such as EWS/pPNET and desmoplatic small round cell tumor, may express, even if less frequently and usually with focal extension, WT1 at the cytoplasmic level. With regard to desmoplastic small round cell tumor, a cytoplasmic WT1 staining can be obtained by using WT1 (N-terminus-clone 6F-H2) antibodies (Nakatsuka et al., 2006; Bisceglia et al., 2011a), while a similar cytoplasmic immunoreactivity has been reported in up 43% or 63% of cases of EWS/pPNET by using antibodies against both Cterminus or N-terminus of WT1 protein, respectively (Nakatsuka et al., 2006). This strongly suggests that a small round cell tumor which expresses a diffuse and strong cytoplasmic WT1 staining is likely to be a rhabdomyosarcoma, but this finding should be interpreted in the context of other antibodies, such CD99, FLI-1, desmin, myogenin, MyoD1, cytokeratins, and INI1 protein. Congential/infantile fibrosarcoma versus other spindle cell tumors There are no specific immunomarkers for congenital/infantile fibrosarcoma, even if this tumor may show focally alpha-smooth muscle actin and/or desmin, suggesting that, at least, a minor component of neoplastic cells is myofibroblastic in nature. Recently, we have shown that congenital/infantile fibrosarcoma exhibits a strong and diffuse cytoplasmic WT1 immunoreactivity by using WT1 N-terminus antibodies (Fig. 7B) (Magro et al., 2014a). Although sensitive, this staining is not specific because other spindle cell lesions, such spindle cell rhabdomyosarcoma, benign and malignant peripheral nerve sheath tumors, leyomyosarcoma, myofibroma/myofibromatosis, are reported to be variably WT1positive (Nakatsuka et al., 2006; Schittenhelm et al., 2010; Singh et al., 2012; Magro et al., 2014a, 2015c). This possibility should be kept in mind by pathologists, especially when dealing with small biopsies containing a spindle cell proliferation. However we suggest that WT1 staining is helpful in confirming the diagnosis of congential/infantile fibrosarcoma in the appropriate clinicopathologic context (tumor localized in the extremities of an infant, highly cellular and composed of interlacing fascicles of uniform spindle cells with only low nuclear atypia, and focal expression of alpha-smooth muscle actin and/or desmin) (Magro et al., 2014a). The morphologically and immunohistochemically ambiguous cases can be correctly diagnosed by means of molecular analyses showing the recurrent translocation t(12;15) (p13;q25) with an ETV6-NTRK3 gene fusion, typically found in most cases of infantile fibrosarcoma (Coffin and Alaggio, 2012). Conclusions WT1 protein is a useful marker for diagnosis of malignant tumors in children and adolescents. However, it should be emphasized that not only Wilms tumor, but also other neoplasms, including desmoplastic small round cell tumor, malignant rhabdoid tumor, can express WT1 protein at nuclear level. Pathologists should be aware of the possibility that nuclear and/or cytoplasmic WT1 immunostaining mainly depends on the tumor examined

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