Prediction of histopathological local staging by radiological findings and differential diagnosis overview in children with nephroblastoma.

BACKGROUND: Nephroblastoma is the most common renal malignancy in children kidney. They are highly heterogeneous tumors with challenging imagistic and histopathological (HP) differential diagnosis. Imaging is critical for understanding local anatomy, staging and for planning surgical approach.
PURPOSE: To determine whether HP staging can be successfully predicted by the imagistic staging using computed tomography. Also, we find it important to make a brief review of the imagistic, HP and immunohistochemical differential diagnosis of nephroblastoma, considering that a correct diagnosis is essential for an appropriate therapeutic strategy in all stages. PATIENTS,
MATERIALS AND METHODS: We present a retrospective study of the medical dossiers of 22 patients that underwent surgery at our Center between 2014 and 2020. We provided descriptive data and compared imagistic and HP staging using a Mann-Whitney U-test. An up-to-date literature review was also done.
RESULTS: We found that imagistic staging tends to under- or over-stage at similar rates and that the difference between the two staging systems is statistically significant. Immunohistochemistry is necessary for establishing the correct diagnosis, especially in cases with one predominant HP pattern.
CONCLUSIONS: HP and imagistic staging are not yet sufficiently similar for successfully predict the former via imagistic means.

⧉ Introduction

Nephroblastoma is a malignant embryonic tumor originating from renal precursor cells, consisting of an inconstant percentage of embryonal renal components: blastema, epithelium and stroma [1,2]. Wilms tumor (WT) is the most frequent renal tumor in children and represents about 6% of all malignancies in childhood [2]. Consecutive clinical trials organized by the National Wilms Tumor Study Group (NWTSG), which was superseded by the Children’s Oncology Group (COG) in 2002, the International Society of Pediatric Oncology (Société Internationale d’Oncologie Pédiatrique – SIOP) and other national study groups established an overall survival rate over 90% [3,4]. The difference between the therapeutic approaches in the NWTSG/COG and SIOP protocols has repercussion for the imaging method used for diagnosis. In the NWTSG/COG protocol, chemotherapy and radiotherapy follows surgical treatment, so imaging methods are used to add accurate anatomical description for staging and devising a personalized surgical strategy [4,5]. Echography is the first chosen imaging method, confirming the existence of a renal tumor without ionizing radiation and allows an initial evaluation of the other kidney [6,7]. However, magnetic resonance imaging (MRI) or computed tomography (CT) are necessary for gaining all information for diagnosis and staging [6]. While imaging [ultrasonography (US), CT, and MRI] can predict up to 95% of nephroblastomas, they cannot predict the histopathological (HP) subtypes and therefore HP examination is mandatory [8]. Accurate HP description and stadialization of these tumors are essential since their treatment and outcomes varies [9].

Aim

The aim of our study was to determine whether HP staging can be successfully predicted by the imagistic staging using CT.

⧉ Patients, Materials and Methods

Patients

We performed a retrospective study using our institutional database to identify patients who underwent surgical treatment for nephroblastoma at our center during a 6-year period, between September 2014 and September 2020. We identified a total of 33 patients initially diagnosed with renal tumors presenting nephroblastoma-like characteristics on CT scans. From this group, we excluded five patients who underwent surgical treatment at a different Center, two patients who had a different HP diagnosis [clear cell renal carcinoma and intrarenal neuroblastoma (NB)], one patient who was treated following SIOP protocol and three patients who were lost to follow-up. A total of 22 patients histopathologically confirmed with nephroblastoma were left to be included in the analysis. Patient demographics, including age, gender, urban or rural environment, associated syndromes, presenting signs and symptoms, oncological and surgical management were determined from a review of each patient’s medical record.

Methods

Thoraco-abdomino-pelvic CT studies were used for staging in all cases. Abdominal US was also used in all cases as the initial diagnostic method. Postoperative local staging was established via HP. CT analysis was performed in a standard fashion, particular attention being paid to tumor size, capsular integrity, invasion of adjacent organs, venous and ureteral extension, nodal involvement, metastases, and status of the contralateral kidney. Imaging staging was performed using the NWTS/COG staging system (Table 1).

Table 1

NWTS/COG staging of nephroblastoma

Stage
I	Tumor is limited to kidney and is completely resected.
	The tumor may be protruding into the pelvic system and “dipping” into the ureter.
	The vessels of the renal sinus are not involved.
	Intrarenal vessel involvement may be present.
II	The tumor extends beyond kidney or penetrates through the renal capsule and/or fibrous pseudocapsule into perirenal fat but is completely resected.
	The tumor infiltrates the renal sinus and/or invades blood and lymphatic vessels outside the renal parenchyma but is completely resected.
	The tumor infiltrates adjacent organs or vena cava but is completely resected.
III	Incomplete excision of the tumor, which extends beyond the resection margins.
	Any abdominal lymph nodes are involved.
	Tumor rupture before or intraoperatively.
	The tumor has penetrated through the peritoneal surface.
	Tumor thrombi present at resection margins of vessels or ureter, transected or removed piecemeal by surgeon.
	The tumor has been surgically biopsied.
IV	Hematogenous metastases (lung, liver, bone, brain, etc.) or lymph node metastases outside the abdominopelvic region.
V	Bilateral renal tumors at diagnosis.

COG: Children’s Oncology Group; NWTS: National Wilms Tumor Study

We followed NWTS/COG protocol that involves upfront nephrectomy when suitable, succeeded by stage-directed therapy. This is different from the SIOP protocol, where nephrectomy is followed by chemotherapy in most children. All children who had resectable tumors were treated with initial surgical excision followed by adjuvant therapy (chemotherapy with or without radiotherapy). For stage I, II and resectable stage III tumors, primary radical nephrectomy was performed. For unresectable stage III tumors, stage IV and stage V tumors, neoadjuvant chemotherapy was administered. Following nephrectomy, all children received chemotherapy. Adjuvant radiotherapy was reserved for children with stage III or above disease.

HP diagnosis was made on routine Hematoxylin–Eosin (HE)-stained slides prepared from surgical excised renal tumors prior or after chemotherapy. Biopsy material was evaluated for predominant HP pattern (blastemal, epithelial, or stromal). A pattern is predominant when more than 65% of the sample is composed of one of these cell types. Risk stratification was established based on these aspects. The presence or absence of anaplasia was documented. Attention was paid to vascular invasion, capsular integrity, intraureteral extension, surgical margins, and lymph node invasion.

Immunohistochemistry was performed as an ancillary technique for establishing a definitive diagnosis in 12 (55%) cases, using standard immunohistochemical (IHC) protocol. Immunohistochemistry using a small panel of markers is useful in establishing a final diagnosis in most patients.

Data analysis

Descriptive data was analyzed using Microsoft Excel. Staging results were statistically analyzed using Statistical Package for the Social Sciences (SPSS) software. All patients were categorized into five classes according to their imagistic staging at the time of diagnosis and to the HP local staging. The two staging distributions were compared using a Mann–Whitney U-test. A level of significance α=0.05 was used.

⧉ Results

We reviewed the records of 22 patients who underwent surgery for nephroblastomas at our Center. The median age at diagnosis was three years ranging from 0 to 11 years. The male to female ratio was 1:1. There were 16 (73%) patients from rural areas and six (27%) patients from urban areas. Associated anomalies were found in only one patient who had Beckwith–Wiedemann syndrome. There were 15 patients that presented with abdominal pain, 14 with palpable abdominal mass, nine with weight loss and six with hematuria. Less frequent manifestations were fever, loss of appetite, vomiting and acute urine retention (Table 2). All patients underwent surgery. Nephrectomy was performed in 20 (91%) patients, and partial nephrectomy in two (9%) patients. One patient required the excision of neighboring organs (sigmoid colon and superior rectum). All patients received adjuvant chemotherapy and seven patients received adjuvant radiotherapy.

Table 2

Demographics and history

Median age	Three years (ranging from 0 to 11 years)
Sex (n, %)	Male	11	50%
	Female	11	50%
Medical history (n, %)	Beckwith–Wiedemann syndrome	1	5%
Presenting symptoms (n, %)	Abdominal pain	16	73%
	Abdominal mass	12	55%
	Weight loss	10	45%
	Hematuria	7	32%
	Fever	6	27%
	Loss of appetite	5	23%
	Vomiting	4	18%
	Acute urinary retention	1	5%
Provenance (n, %)	Rural	16	73%
	Urban	6	27%

n: No. of cases

Sex (

Medical history (

Presenting symptoms (

Provenance (

Radiological staging

Only one patient (4.5%) in our study group had bilateral nephroblastoma. Vascular invasion was objectified in four (18.1%) cases, renal pelvis or ureteral invasion in five (22.7%) cases, capsular effraction in five (22.7%) cases and adjacent organ invasion in four (18.1%) cases, lymphadenopathies in 10 (45.4%) cases and metastases in five (22.7%) cases. Most tumors were classified as stage III (40.9%), followed by stage II (27.2%) and stage IV (55.7%) based on imaging studies. Only one patient (4.5%) was classified as stage I (Figure 1).

Figure 1

Frontal and transversal computed tomography (CT) sections of different nephroblastoma stages

HP staging and IHC

Renal tumors from 22 children treated at our Institution between 2014 and 2020 were histopathologically studied. Only one case (4.5%) was biopsied prior to chemotherapy. In all other cases the entire tumor was received as a resection specimen. Neoadjuvant chemotherapy was administrated in eight (34.7%) cases, for stage III unresectable tumors, stage IV and V tumors. Post-chemotherapy tumor necrosis was described in only one case (4.5%). A predominant HP pattern (Figure 2a,2b,2c,2d) was found in four (18.1%) tumors. Three (13.6%) tumors had a biphasic pattern and the majority, 14 (63.6%), had a triphasic pattern (Figure 3a,3b,3c,3d,3e). In all monophasic tumors, blastemal cells were predominant. All biphasic tumors had epithelial and blastemal elements. Only one tumor had focal anaplasia. Based on predominant cell types, tumors were classified into low, intermediate, or high risk. Most patients had an intermediate risk, 13 (59%), while low risk was attributed to only three (6.6%) patients and high risk to six (27.2%) patients (Figure 4).

Figure 2

Blastemal, epithelial and stromal component: (a) Mesenchymal component represented by loose connective tissue and small vessels; (b) Blastemal component formed by small ovoid cells with reduced eosinophilic cytoplasm and hyperchromatic nuclei showing high mitotic rate; (c) Epithelial component forming closely packed rosettes, resembling neuroectodermal tissue; (d) Biphasic pattern in nephroblastoma showing epithelial component forming small tubules and surrounded by mesenchymal component. Hematoxylin–Eosin (HE) staining: (a and d) ×100; (c) ×200; (b) ×400

Figure 3

Biphasic and triphasic pattern of nephroblastoma: (a) Triphasic pattern in nephroblastoma with micronodules of blastema (bottom left), epithelial component forming tubules and immature glomeruli, connected by connective tissue with variable fibrotic stroma; (b) Triphasic pattern in nephroblastoma depicting islets of blastema (bottom field), small tubules (central) embedded in a loose connective tissue; (c) Biphasic pattern depicting well-formed tubules and incomplete glomeruli in the epithelial component (center), enclosed by the blastemal tissue; (d) Triphasic nephroblastoma with intermingled elements; (e) Triphasic nephroblastoma illustrating blastema (right) mixed with tubules and immature glomeruli and loose mesenchyme (left). HE staining: (a–c) ×100; (d) ×50; (e) ×200

Figure 4

Histopathological (HP) risk stratification

Immunohistochemistry was performed in 12 (55%) cases (Figure 5a,5b,5c,5d). Ki67 proliferation index was calculated. It ranged from 20% to 90%, with the highest value in stage II – 70%, in stage III – 75%, and in stage IV – 90%. A panel of markers were used to establish the differential diagnosis according to the predominant HP pattern. In our analysis, all monophasic nephroblastoma had a blastemal predominant pattern, and in all cases, immunohistochemistry markers were used to obtain a certain diagnosis, including WT1, pan-cytokeratin (CK) AE1/AE3, desmin and paired-box 8 (PAX8). In all cases, immunohistochemistry confirmed the diagnosis of monophasic nephroblastoma.

Figure 5

Immunohistochemical patterns: (a) IHC for Ki67 showing positivity in approximately 75% of tumor cells (×100); (b) IHC for WT1 showing positivity in blastemal tissue (×100); (c) IHC for high-molecular pan-CK AE1/AE3 in epithelial elements (×100); (d) IHC for vimentin showing positive expression in blastemal and mesenchymal elements and negative reaction in epithelial tissue (×100). CK: Cytokeratin; IHC: Immunohistochemistry; WT1: Wilms tumor 1

Various elements were examined to establish correct staging of nephroblastomas: renal capsule involvement, ureteral extension, vascular invasion, lymph node involvement and surgical margins status (Figure 6a,6b,6c,6d). Capsular effraction was seen in 10 (45.4%) patients, renal pelvis or ureteral extension in six (27.3%) patients, microvascular invasion in 11 (50%) patients and lymph node involvement in 10 (45.4%) patients. Most tumors, 19 (86.3%), had clear resection margins, while in three (13.7%) cases complete excision was not achieved. Most of the tumors were classified as stage III (40.9%), followed by stage II and stage IV (27.2% each). Two children had stage I nephroblastoma (9%). Only one patient had bilateral nephroblastoma – stage V (4.5%) (Table 3).

Figure 6

Histopathological aspect of lymphovascular, sinus and capsular invasion: (a) Necrotic area showing cellular and nuclear debris in an aggressive nephroblastoma, blastemal-dominant; (b) Vascular invasion showing blastemal embolus; (c) Tumor invasion into the fibrous capsule, below the upper pole (adrenal tissue-upper field); (d) Vascular invasion showing blastemal embolus. HE staining: (a, b and d) ×100; (c) ×25

Table 3

Imagistic and HP staging criteria

Criteria	CT (% of cases)	Histopathology (% of cases)
Vascular invasion	18.1%	50%
Basinetal/ureteral invasion	22.7%	27.3%
Capsular effraction	22.7%	47.5%
Lymph node invasion	45.4%	45.4%

CT: Computed tomography; HP: Histopathological

Radiological and HP staging correlation

In a review of the records of all children confirmed with nephroblastoma that underwent surgery at our Center, we found 22 patients that were classified into five categories according to their imagistic and HP stage. We found one patient with stage I, six with stage II, eight with stage III, four with stage IV and one with stage V nephroblastoma, according to their imagistic exams. According to the HP staging, we found two patients with stage I, five with stage II, eight with stage III, four with stage IV and one with stage V (Figure 7).

Figure 7

Imagistic and histopathological (HP) stage correlation

We found four patients without correlation between imagistic and HP staging. Two of these patients had an imagistic stage II but HP stage III, one had an imagistic stage III, but HP stage II and one had imagistic stage III and a HP stage I (Table 4).

Table 4

HP staging compared to CT staging (I–V)

HP staging	CT I (n)	CT II (n)	CT III (n)	CT IV (n)	CT V (n)	Total
I	1	0	1	0	0	2
II	0	4	1	0	0	5
III	0	2	7	0	0	9
IV	0	0	0	5	0	5
V	0	0	0	0	1	1
Total	1	6	9	5	1	22
	concordance			discordance

CT: Computed tomography; HP: Histopathological; n: No. of cases

CT I (

CT II (

CT III (

CT IV (

CT V (

⧉ Discussions

Imagistic techniques

Imagistic evaluation for renal tumors should begin with abdominal US, that can establish if the tumor is intra- or extrarenal and if its predominant component is dense or cystic [7, 10]. We performed abdominal US in all cases as the initial imagistic method. On US, nephroblastoma usually appears as a large intrarenal mass that shows heterogeneous echotexture, well-defined margins, delimited by a pseudocapsule [5, 11]. Nephroblastomas can have extensive hypoechoic regions representing central necrosis or cyst development. Hyperechoic regions can be areas of fat, calcification, or hemorrhage. It can also appear as a solid round mass, less frequent [4,5]. US is helpful for the establishment of caval permeability and inferior vena cava tumor thrombus [12,13]. We identified one case of nephroblastoma with intravenous extension visible on US, in a two-year-old child. Because US is operator dependent and regarding the limited field of vision, supplementary imaging methods for extended characterization and precise staging are necessary [7].

In a few cases, nephroblastoma can be predominantly cystic, called cystic partially differentiated nephroblastoma, and can be hard to differentiate from other cystic renal tumors, benign and malignant [14,15,16]. We identified one case of a 2-year-old boy who presented for fever and vomiting. In imaging studies, two round, well defined lesions with fluid density were identified in the right kidney. The initial clinic and imagistic diagnosis were renal abscess. Intraoperatively two well defined, encapsulated tumors that had an elastic consistency and white color were identified. Lesion excision was performed. HP examination established the diagnosis of biphasic nephroblastoma. Postoperative chemotherapy was administered.

CT of the abdomen with administration of intravenous iodinated contrast is also used for diagnosis and evaluation of nephroblastomas [14, 17,18]. We performed CT scans as the main imagistic method for preoperative staging in all patients. On CT, nephroblastoma is described as a heterogeneous soft-tissue density tumor with less enhancement compared to the adjacent renal tissue [19]. Nephroblastomas often contain hypodense regions because of necrosis, hemorrhage or cysts, but fat and calcifications can be seen as well [20,21]. More CT aspects can comprise of infiltration or distortion of the pyelocalyceal system, vascular extension or lymph node involvement [17]. CT allows appropriate staging of the disorder and identifies the existence of nephrogenic rests (NR) in the renal tissue [13, 15]. Abdominal CT can identify the existence of tumor rupture, representing an important risk factor for recurrence of the disease [2]. Less characteristic imagistic aspects are perirenal bleeding that can exist without identifying a tumor rupture, retroperitoneal liquid and intra-abdominal peritoneal effusion [15].

Initially, the differential diagnosis of nephroblastoma is done with extrarenal intra-abdominal tumors. When the renal origin of the tumor is settled, other disorders to be included in the differential diagnosis of nephroblastoma are congenital mesoblastic nephroma, clear cell sarcoma of the kidney (CCSK), malignant rhabdoid tumor of the kidney and renal cell carcinoma (RCC), also known as non-WTs. Non-WTs are frequently identical with nephroblastoma on imaging studies [15, 21].

Congenital mesoblastic nephromas are histopathologically divided into classical and cellular subtypes. On CT scans, the classical subtype looks like a homogeneous solid tumor and the cellular form is heterogeneous with regions of necrosis, hemorrhage, and cystic degeneration [22].

RCC appears smaller on CT compared to nephroblastoma, enlarges progressively and may penetrate neighboring structures altering normal kidney architecture [23]. In comparison with nephroblastoma, RCC is frequently bilateral, and calcifications are often found [16]. We identified one case of an 8-year-old girl who presented with a massive, palpable abdominal tumor. Abdominal US was performed, identifying a right renal tumor. Abdominal MRI was performed for supplementary characterization of the tumor and identified a large (117/115/151 mm) renal tumor, with hyperintensity on T2-weighted images, compatible with renal nephroblastoma. Preoperative chemotherapy was administered, according to SIOP-2001 protocols for nephroblastoma, followed by nephrectomy. HP studies in association with IHC analysis established the diagnosis of clear cell renal carcinoma with alveolar soft part sarcoma locus (ASPL)–transcription factor binding to immunoglobulin heavy constant mu (IGHM) enhancer 3 (TFE3) translocation. Surgical excision was followed by adjuvant chemotherapy and radiotherapy.

HP and IHC overview

Typically, nephroblastomas comprise of three HP components, which are the basis for the differential diagnosis: mesenchymal elements resembling primitive fetal mesenchyme (blastema), epithelial elements that recall fetal renal tubules and glomeruli and blastomatous elements representing clusters of blast cells that are involved in the coinage of the term nephroblastoma [9, 24]. The amplitude and the degree of maturation of these elements vary considerably, resulting in a distinguishable HP appearance of each tumor [25].

The diagnosis is usually not problematic in triphasic or even biphasic nephroblastoma, but if only one element is found, particularly in a small biopsy sample, the differential diagnosis can be difficult [26]. In our study, we identified four cases of monophasic pattern, all of them having blastemal predominance and three cases of biphasic pattern. IHC is helpful in reaching a definitive diagnosis in most cases. Different IHC panels are indicated based on the HP predominant pattern (Table 5) [26].

Table 5

IHC panels for differential diagnosis of pediatric renal tumors

Patterns	Panels
	Recommended panel		Additional panel
Epithelial pattern	WT1	CK7	PAX8	INI1
	Pan-keratin	TFE3*	BCOR*	CD10
	Melanocytic markers	TFEB*	Cathepsin K	Vimentin
	AMACR	BRAF V600E*	ALK	Cyclin D1
			2SC/FH
Stromal pattern	WT1	BCL-2	INI1	pan-Trk*
	CD34	NGFR*	CD99	TLE1*
	Cyclin D1	BCOR*	BRAF V600E*
Blastemal pattern	WT1	NE markers	CD56	BCOR*
	INI1	Cyclin D1	CD99	PHOX2B*
	CKs	NB84*	CD45
	Desmin	NGFR*

2SC/FH: S-(2-succinyl) cysteine/Fumarate hydratase; ALK: Anaplastic lymphoma kinase; AMACR: Alpha-methylacyl-coenzyme A (CoA)-racemase; BCL-2: B-cell lymphoma 2; BCOR: B-cell lymphoma 6 (BCL-6) corepressor; BRAF: V-Raf murine sarcoma viral oncogene homolog B1; CD: Cluster of differentiation; CK7: Cytokeratin 7; IHC: Immunohistochemical; INI1: Integrase interactor 1; NB84: Neuroblastoma 84; NE: Neuroendocrine; NGFR: Nerve growth factor receptor; PAX8; Paired-box 8; PHOX2B: Paired-like homeobox 2b; TFE3: Transcription factor binding to immunoglobulin heavy constant mu (IGHM) enhancer 3; TFEB: Transcription factor EB; TLE1: Transducin-like enhancer of split 1; Trk: Tyrosine receptor kinase; WT1: Wilms tumor 1. *If available

Ki67, a tumor proliferation marker with prognostic significance, is evaluated in nephroblastomas. High Ki67 proliferation index is associated with more aggressive clinical behavior and is proved to be an important determinant of distant metastasis and tumor related deaths [27].

Blastema is the least differentiated and hypothesized the most malignant element [25]. It is composed of small blue cells with overlapping nuclei and brisk mitotic activity. Primitive tubular epithelial elements can be found in the center of blastemal nodules and morphologically mimic NB-like areas with pseudorosettes [28].

Pure blastemal type nephroblastoma may be difficult to distinguish from other embryonal small round blue cell tumors, including NB, malignant rhabdoid tumor of the kidney, primitive neuroectodermal tumor, Ewing sarcoma (EWS), desmoplastic small round cell tumor (DSRCT) and lymphoma [24, 26]. To reach the correct diagnosis in cases of difficult differential diagnosis it is essential to use immunohistochemistry and molecular biology in search for distinctive elements (Table 6) [29]. The blastemal elements of nephroblastoma present nuclear WT1 positivity in 80% of cases [30].

Table 6

IHC characteristics of the most common renal tumors with a blastemal pattern

Blastemal pattern	Immunohistochemistry
	WT1(n)	INI1	NGFR	Keratin	CD99	NE	Desmin	PAX8	NB84	Cyclin D1
WT	+	++	-	+/-	-	--	-	++	--	-
MRTK	-	--	-	+/-	+/-	NA	+/-	--	NA	++
NB	-	++	+	-	--	++	--	--	++	+
DSRCT	+	++	--	+	-	--	++	--	-	-/+
EWS	--	++	-	-	++	--	--	--	+/-	++

CD99: Cluster of differentiation 99; DSRCT: Desmoplastic small round cell tumor; EWS: Ewing sarcoma; IHC: Immunohistochemical; INI1: Integrase interactor 1; MRTK: Malignant rhabdoid tumor of the kidney; n: Nuclear; NA: Not available; NB: Neuroblastoma; NE: Neuroendocrine; NGFR: Nerve growth factor receptor; PAX8: Paired-box 8; WT: Wilms tumor. Scoring: ++ >95% positive cases; + 76–95% positive cases; +/- 51–75% positive cases; -/+ 26–50% positive cases; - 5–25% positive cases; -- <5% positive cases

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DSRCTs share several IHC characteristics with blastemal-type nephroblastoma, but are rare and the diagnosis should be established only if genetic tests identify EWS–WT1 t(11;22)(q13;q12) translocation [17, 24]. DSRCT are WT1-positive but only with the C-terminal clone of WT1, and nephroblastomas are positive with both the N-terminal and C-terminal clone. DSCRT also show CK and desmin positivity. PAX8 is negative in DSCRT [29]. In our study group, we identified one case of a six-year-old boy radiologically diagnosed with three intra-abdominal heterogeneous masses, the larger one localized in the left hemiabdomen (98/90/128 mm) and the other two in the pelvic region and an intrathoracic tumor with similar characteristics. The initial radiological suspicion was of NB. A biopsy was performed, and HP examination identified a malignant DSRCT. HP and IHC examination of the entire tumor after nephrectomy established the diagnosis of biphasic nephroblastoma with 95% blastemal cells and 5% epithelial component.

NB presents with high levels of catecholamines and on HP exam its cells present non-overlapping nuclei and coarse salt and pepper chromatin [28]. Both tumors can be positive for neuron-specific enolase (NSE) and cluster of differentiation (CD)56, but WT1 marker is negative in NB and NB84a is negative in nephroblastoma [26]. NB is also positive for synaptophysin, chromogranin A and paired-like homeobox 2b (PHOX2B). NB is PAX8-negative [30].

The epithelial component may contain the entire spectrum of differentiation from early stages of tubular formation with primitive epithelial rosette-like structures to differentiating tubules or glomeruli-like elements, showing different moments of nephrogenesis [23, 26, 28].

Pure epithelial type of nephroblastoma may be difficult to distinguish from hyperplastic perilobar NR, metanephric adenoma (MA) and RCC [26]. Immunohistochemistry is useful in establishing a final diagnosis in most cases (Table 7) [29,30].

Table 7

IHC characteristics of the most common renal tumors with an epithelial pattern

Epithelial pattern	Immunohistochemistry
	WT1	AMACR	Pan-keratin	CK7	TFE3	TFEB	Melanocytic markers	BRAF V600E
WT	++	--	+/-	--	--	--	--	--
NR	+	NA	+	-/+*	NA	NA	NA	-
MA	++	-	+/-	-/+*	NA	NA	NA	+
Xp11.2 tRCC	--	++	-/+*	-	++	--	-/+	--
t(6,11) RCC	--	+	-	-	--	+	+	--
PRCC	--	++	++	+	--	--	--	--

AMACR: Alpha-methylacyl-coenzyme A (CoA)-racemase; BRAF: V-Raf murine sarcoma viral oncogene homolog B1; CK7: Cytokeratin 7; IHC: Immunohistochemical; MA: Metanephric adenoma; NA: Not available; NR: Nephrogenic rest; PRCC: Papillary renal cell carcinoma (RCC); TFE3: Transcription factor binding to immunoglobulin heavy constant mu (IGHM) enhancer 3; TFEB: Transcription factor EB; tRCC: Translocation associated RCC; WT: Wilms tumor. *If positive, often only focally. Scoring: ++ >95% positive cases; + 76–95% positive cases; +/- 51–75% positive cases; -/+ 26–50% positive cases; - 5–25% positive cases; -- <5% positive cases

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Highly differentiated epithelial-type nephroblastoma can be formed of small, well-differentiated and closely packed tubules resembling MA, which consists of small, uniform, closely packed tubules and papillae, but the last one can be identified by the absence of capsule between the tumor and renal parenchyma, well defined delimitation from the renal parenchyma, the identification of small uniform cells with scant cytoplasm, bland nuclei, fine chromatin, the lack of mitotic activity, atypia and psammoma bodies [26, 31,32]. MA also has an IHC pattern: CK7-negative, alpha-methyl-acyl-coenzyme A (CoA)-racemase (AMACR)-negative, WT1-positive, CD57-positive. The majority of MA carry a V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation (BRAF V600E most common), that can be demonstrated by immunohistochemistry. Nephroblastomas and MAs with overlapping HP and molecular characteristics were reported [23].

NR are foci of embryonal cells that remain after 36 weeks of gestation and are considered predecessor of nephroblastomas [24, 33]. Hyperplastic NR may be hard to differentiate from nephroblastomas because of the lack of HP, IHC or molecular criteria that can be used [17, 19, 23].

The stromal component can consist of densely packed undifferentiated mesenchymal cells or loose cellular myxoid areas. Heterologous differentiation of neoplastic stroma in the form of well differentiated smooth or skeletal muscle cells, fat tissue, cartilage, bone and even glial tissue is present in some cases, mostly in tumors that have undergone preoperative chemotherapy [29, 33].

In the differential diagnosis of pure stromal type nephroblastoma, the following should be considered: CCSK, congenital mesoblastic nephroma and metanephric stromal tumor (MST) [26, 28]. Even though some renal masses with a mesenchymal pattern miss a specific IHC profile, a group of markers may be useful to distinguish the tumors (Table 8) [29]. In the stromal component of nephroblastoma, WT1 is often weak or absent [29].

Table 8

IHC characteristics of the most common renal tumors with a stromal pattern

Stromal pattern	Immunohistochemistry
	WT1	NGFR	BCOR	Cyclin D1	INI1	CD34	BCL-2
WT	+/-	-	-/+*	-	++	-/+	+
CSSK	--	++	+	++	++	--	+/-
Cellular/Mixed CMN	--	-*	-*	+/++	++	--	--
Classic CMN	--	--	-*	+/++	++	--	--
MST	+/-	-	-*	NA	++	++	NA

BCL-2: B-cell lymphoma 2; BCOR: B-cell lymphoma 6 (BCL-6) corepressor; CCSK: Clear cell sarcoma of the kidney; CD34: Cluster of differentiation 34; CMN: Congenital mesoblastic nephroma; IHC: Immunohistochemical; INI1: Integrase interactor 1; MST: Metanephric stromal tumor; NA: Not available; NGFR: Nerve growth factor receptor; WT: Wilms tumor. *If positive, often only focally. Scoring: ++ >95% positive cases; + 76–95% positive cases; +/- 51–75% positive cases; −/+ 26–50% positive cases; - 5–25% positive cases; -- <5% positive cases

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CCSK is classically formed of bland ovoid cells with monomorphic nuclei with finely dispersed chromatin and small nucleoli organized in cords and nests disconnected by a chicken-wire vasculature, but there can be different patterns leading to an increased number of misdiagnoses [10, 29]. CCSK shows a distinctive IHC profile: cyclin D1, B-cell lymphoma 6 (BCL-6) corepressor (BCOR), nerve growth factor receptor (NGFR)-positive and WT1-negative [30].

Nephroblastomas with diffuse anaplasia or blastemal predominance are consecrated as high-risk tumors and need aggressive treatment [24]. We identified one case of focal anaplasia in a 3-year-old girl who presented with pulmonary metastases at diagnosis. Anaplastic nephroblastomas are defined by the presence of large, atypical multipolar mitotic figures and significantly enlarged and hyperchromatic nuclei. Anaplasia may be focal or diffuse. Anaplasia is culpable for adverse response, mostly in advanced tumor stages [32,33].

Preoperative chemotherapy may highly disturb the primary histopathology by reducing or enhancing some elements or by activating maturation [34,35].

Clinical studies show that the results of patients with nephroblastomas are reliant to HP description [29]. Favorable histopathology is defined by the presence of all three HP components and the lack of diffuse anaplasia [16]. There are three HP risk groups: low risk (completely necrotic nephroblastoma or cystic partially differentiated nephroblastoma), intermediate risk (regressive, epithelial, stromal, mixed, or focal anaplastic nephroblastoma) and high risk (blastemal or diffuse anaplastic nephroblastoma) [4, 29]. We identified three patients with favorable histopathology or low risk, 13 patients with intermediate risk and six patients with unfavorable histopathology or high risk. All three HP components of nephroblastomas present individual proliferation potential and respond differently to therapy [10].

Radiological and HP staging and correlation

Staging criteria for nephroblastoma are based entirely on the anatomic extension of the mass, without taking into consideration genetic, biological, or molecular markers [14]. Stage is one of the most relevant therapeutic and prognostic criteria for kidney masses [4]. Staging is an important issue in multiple centers because kidney tumors are often large and usually it is hard to establish their relationship with normal anatomical elements [16].

Imagistic staging of nephroblastomas encompasses several steps. The radiologist needs to establish if the mass is limited to the kidney and if it can be totally removed with the surface of the renal capsule intact. In this case, the patient is regarded as stage I [36,37]. Development of the mass outside the renal capsule obviates the classification of stage I [4]. Local extension into the neighboring tissues, renal vessels or inferior vena cava outside the kidney can be identified and, when grossly removed, the child is classified as stage II [12, 20]. The regional lymph nodes must be defined because this automatically places the patient into stage III. Lymph node status may be difficult to appreciate in large tumors obscuring lymph nodes or in case of lymph nodes larger than 1 cm that appeared secondary to the tumor, rather than infiltration [38]. Also, lymph nodes smaller than 1 cm can be infiltrated and not identified as atypical by radiological methods [15]. In the NWTS/COG protocols, staging includes intraoperative lymph node sampling because intraoperative macroscopic evaluation of positive ganglia is not reliable, false negative rates being as high as 31.3% [38]. Accurate interpretation of the emerging imaging data (tumor limited to the kidney, tumor extended beyond the kidney and lymph node involvement) permits the patient to be correctly classified into stages I–III [39]. Patients who present with systemic metastases usually in the lung, liver, bone or brain are classified as stage IV [14, 40]. Of these sites, the lung remains the most common, followed by the liver. It is critical to differentiate between lung nodules and atelectasis at the base of the lungs. Children with bilateral renal tumors at the diagnosis are classified as stage V. This is found in 5–7% of patients [2, 10]. The high-risk groups for bilateral nephroblastoma are those children who have multifocal lesions in their index kidney, children with nephroblastomatosis, those with associated congenital anomalies and children with a family history of nephroblastoma [4, 10].

At the moment, there are two important HP staging systems that are applied: an upfront surgery-based system developed by NWTSG, and an upfront chemotherapy-based system developed by SIOP [16, 39]. Both staging systems have proven valuable in predicting outcomes [10]. Even if the architecture of nephroblastomas varies significantly, the tumor is allocated a HP type if more than two-thirds of the tumor tissue is of one element [4, 21]. From a review of NWTSG studies, favorable and unfavorable HP subtypes are defined as the presence or the lack of anaplasia [16, 41]. The existence of anaplasia has been attributed to low tumor responsiveness to adjuvant chemotherapy [16]. The definition of focal anaplasia is based on topographical principles, requiring that anaplasia be limited to just a few specific areas [10, 35]. Also, the identification of HP subtypes is important, as risk stratification is also established by it [32, 42].

In our study, imagistic staging corresponded with HP staging in 82% of the patients. This number is comparable to the literature [38] and has been increasing with the improvement of imaging techniques [2], however CT scans are not yet sufficiently free of error to be able to correctly predict HP staging [43].

Other studies show various degrees of correlation between CT staging and HP staging of nephroblastoma, some being as low as 38% correspondence between the two [38]. In our study, the CT examination under staged two of nine (22%) local spread tumors (stage III), over staged one of two (50%) localized renal disease (stage I) tumors and over staged one of five (20%) local extension (stage II) tumors. Under staging tumors might be more significant, especially for stage III tumors, since complete excision is not achieved, therefore the patient has a significantly different prognosis [44].

We performed a Mann–Whitney U-test to evaluate the probability that staging discrepancies could be attributed to chance. The z-value was calculated to be 2.31, which is well over the 1.96 mark for a 5% α-value. This means that we can reasonably state that radiological evaluation of nephroblastoma cannot yet reliably predict HP staging.

⧉ Conclusions

Our study reviewed the medical records of 22 patients diagnosed with nephroblastoma between 2014 and 2020. This group provided a series of descriptive statistics of the demographics, imagistic and HP characteristics for the analysis. We found that the HP staging was over evaluated in 22% of cases and under evaluated at the same rate by the imagistic staging system. We compared the imagistic and HP staging distributions via a Mann–Whitney U-test and concluded that imagistic staging cannot be yet used to predict HP staging with satisfactory precision. Differential diagnosis is challenging especially in cases of monophasic pattern nephroblastomas, immunohistochemistry having a pivotal role for correct diagnosis and subsequent therapeutic management.

Conflict of interest

The authors declare that they have no conflict of interests.