Bilateral Nephroblastic Tumors and a Complex Renal Vascular Anomaly in a Patient With a Mosaic RASopathy: Novel Histopathologic Features and Molecular Insights

Background

Mosaic RASopathies are an emerging group of disorders characterized by mosaic or post-zygotic activating mutations in genes of the RAS/MAPKinase signaling pathway, including KRAS, NRAS and HRAS. This pathway, in concert with PI3K-AKT signaling, controls cell proliferation and differentiation, regulates angiogenesis, and also is critical in oncogenesis. The mutational spectrum in somatic RASopathies differs from classical (germline) RASopathies, and cancer-associated variants are more common. ¹ The phenotype of somatic RASopathies is highly variable, ranging from limited or localized forms to cases with a syndromic presentation and extensive, multi-organ involvement. There is considerable overlap among the different RASopathies, and with other mosaic disorders, including the PIK3CA-related overgrowth spectrum (PROS) caused by PI3K-AKT pathway mutations. ² As a result, diagnostic distinction from PROS disorders such as CLOVES (congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies) can be challenging when there is overgrowth and cutaneous anomalies. Factors thought to determine the phenotype in mosaic disorders include the timing of the mutation during the embryonic development, the cell type affected, the frequency of the allelic variant, the functional impact of the variant, and secondary acquired variants as well as epigenetic and constitutional modifiers.^1,3

Although several reports document malignancies, specifically rhabdomyosarcoma and transitional urothelial carcinoma, in children and young adults with somatic RASopathies,^1,4,5 the lifetime cancer risk in these patients, and the mechanisms through which malignancies develop, remain largely unknown.

We report the renal pathological and molecular findings in a 22-month-old boy with a complex unilateral renal vascular anomaly and multiple bilateral nephroblastic tumors associated with a somatic oncogenic KRAS p.G12D variant and syndromic presentation. Clinical features included a large unilateral epidermal nevus, unilateral lower limb overgrowth and skeletal anomalies, which overlap with other mosaic disorders, notably PROS/CLOVES.

Case Report

The patient was born at term after an uneventful pregnancy. At birth, a large right-sided epidermal nevus extending from the neck to the foot was noted. At 3 months, his right lower limb appeared enlarged with an overlying reticulated capillary malformation. X-rays at 19 months demonstrated overgrowth and angulation of the right tibia and fibula. Also, multiple cystic bone lesions with surrounding osteosclerosis were noted in the right femur, tibia and fibula (Figure 1). No other skeletal anomalies were noted.

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Figure 1.

Large right-sided epidermal nevus extending to the lower extremity at 7 months (A). Right distal lower extremity radiograph at 19 months demonstrating multiple ill-defined, non-expansile lytic lesions with adjacent sclerosis in the distal femur, tibia and fibula. There is diaphyseal, metaphyseal and epiphyseal involvement and associated bowing deformity of the tibia and fibula (B).

Molecular genetic testing identified a KRAS p.G12D oncogenic hotspot variant in the epidermal nevus, but not the blood, consistent with somatic mosaicism. No mutations were found in PIK3CA, FGFR3, or any of the pediatric cancer genes tested. There was no family history of malformations, genetic disorders or pediatric malignancies.

A renal ultrasound at 6 weeks showed a small, non-functioning right kidney, and scattered small cysts in the left kidney. An MRI at 6 months revealed multiple bilateral homogeneous echogenic lesions suspicious for nephroblastomatosis and Wilms tumor (WT). The lesions showed progressive enlargement, and a biopsy at 21 months showed proliferative nephroblastic lesions in both kidneys, though definite distinction between hyperplastic nephrogenic rests (NRs) and WT was not possible. After neoadjuvant chemotherapy, a right-sided nephrectomy and resection of three left-sided lesions were performed.

The nephrectomy specimen (Figure 2) demonstrated three discrete thinly-encapsulated nodules with focal necrosis that were composed predominantly of well-differentiated nephrogenic epithelium showing a mainly solid, expansile, and confluent growth pattern. In addition to scattered foci of nephrogenic epithelium, consistent with perilobar NRs and more discrete non-encapsulated multi-nodular adenomatous NRs, the entire residual kidney showed involvement by a complex vascular anomaly (VA) that extended into the copious perirenal and hilar fat (Figure 3). The VA comprised large, unpaired and thick-walled malformed veins with focal intramural capillary proliferations, patchy myxoid change, foci of chronic inflammation, and occasional thrombi. In addition, it contained nodules of angulated thin-walled veins, focal patchy capillary proliferations surrounded by a fibromyxoid stroma, and occasional clusters of lymphatic vessels.

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Figure 2.

Macroscopic appearances of the right kidney with encapsulated Wilms tumors (A) and the resected tumors from the left kidney (B and C). Microphotographs of right (D and F) and left kidney (E and G) illustrating encapsulated Wilms tumors (star), adenomatous (ANR) and dispersed nephrogenic rests (NR), and vascular anomaly with thick-walled vein (arrows).

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Figure 3.

Complex vascular anomaly (VA), right kidney, with large malformed veins with myxoid change and intramural capillary proliferations (A), clusters of smaller veins and capillaries (B), veins and occasional lymphatics (C) and involvement of a large nerve (D).

The three encapsulated nodules from the left kidney were comparable to the right, though no necrosis was demonstrated (Figure 2). The sparse surrounding renal tissue contained foci of nephrogenic epithelium, smaller non-encapsulated adenomatous NRs, and normal parenchyma. External review favored a diagnosis of WT for the encapsulated nodules in both kidneys, largely based on the solid expansile growth pattern. Otherwise, the cytological and architectural findings overlapped considerably with those of the adenomatous NR.

On molecular testing, using the Oncomine Childhood Cancer Research Assay (ThermoFisher Scientific) and targeted dPCR, the mosaic KRAS p.G12D variant was present at a high allele frequency (40-60%) in the abnormal right kidney, implying it was present in nearly all cells. It was seen at similar high frequencies in the background right renal parenchyma which appeared malformed, dispersed and adenomatous NRs, and bilateral encapsulated nodules, but only at 3-4% in the normal parenchyma of the left kidney (Figure 4). While the intrarenal portion of the VA also shared a high allele frequency (46%), it was lower (10-11%) in the extrarenal portion due to abundant perirenal adipose tissue. Interestingly, two encapsulated nodules of tumor in the right kidney harbored a second FBXW7 p.R479G cancer hotspot variant (40-44%) in nearly all cells. NRs within one of these nodules were negative for this variant. A third tumor nodule on the right and one from the left kidney that were morphologically similar lacked the FBXW7 variant.

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Figure 4.

KRAS p.G12D and FBXW7 p.R479G variant allele frequency (VAF) in Wilms tumors, right (A) and left kidney (C), Wilms tumor associated nephrogenic rest (NR) in right kidney (B), adenomatous NR (D), intra renal (E) and extra renal (F) vascular anomaly, and normal left kidney (G).

Discussion

This case further expands the clinical phenotype of somatic RASopathies, as it is the first to document nephrogenic tumors (NRs and WT) and/or a complex renal VA in a patient with a genetically confirmed somatic RASopathy. Moreover, it illustrates the utility of molecular testing, which ensured accurate classification of this patient’s condition, with a potential for targeted therapy. ⁶

The clinical presentation with a unilateral epidermal nevus, asymmetric limb overgrowth, capillary malformation and bilateral Wilms tumors initially suggested a diagnosis of CLOVES, although there was no lipomatous hyperplasia, making this diagnosis less likely. Also, the cystic bone lesions with peripheral sclerosis in the right lower limb were distinct from the overgrowth and/or VA-associated skeletal anomalies described in CLOVES. ² While the exact etiology is unknown, they may represent multifocal giant cell-rich bone lesions or non-ossifying fibromas which have been described in other RASopathies such as oculoectodermal syndrome, ⁷ albeit the lesions in our case were less expansile and showed more extensive epiphyseal involvement, or dysostotic lesions similar to those described in melorheostosis, another RASopathy. ⁸

PIK3CA and FGFR3 mutations each account for about 30% of epidermal nevi, whereas KRAS mutations are less common.^1,9 Similarly, somatic PIK3CA mutations are commonly identified in a range of vascular anomalies, including CLOVES, fibro-adipose vascular anomaly (FAVA), and sporadic capillary and lymphatic malformations,^1,10 whereas pathogenic RAS/MAPK variants are far less common in sporadic V,^6,11,12 and only rarely documented in somatic RASopathy patients. ¹³

The VA was likely congenital, as the kidney was found to be small and non-functional shortly after birth. The histopathological features were distinct from the rare documented cases of renal VA, which include congenital hemangioma like capillary proliferations, arteriovenous, lymphatic and capillary-venous malformations. ¹⁴ In contrast to those lesions, the vascular anomaly in our case diffusely involved the kidney, disrupted the normal renal architecture, and also extended into the perirenal fat. Its microscopic appearances were distinct from a common venous malformation, which typically shows more extensive clustering of veins and lacks a lymphatic component, but did bear some resemblance with FAVA ¹⁵ ; though to our knowledge FAVA has not been described in the kidney or retroperitoneum, nor have renal VAs been described in somatic overgrowth disorders. While we did not identify a secondary PIKC3CA variant, this does not exclude a functional role of the PI3K/AKT signaling pathway in the development of this complex vascular anomaly.

Nephroblastomatosis and WT have been reported in a small number of patients with PROS,^2,16 though it is not clear if WT development in PROS is driven by secondary mutations as PIK3CA variants are not associated with sporadic WT. The limited evidence for a role of KRAS in the development of Wilms tumor is mainly derived from a mouse model; it was shown that coordinated activation of Ras and β-catenin led to the development of Wilms-like tumors, and was dependent on PI3K/AKT signaling.^17,18 Studies in humans have identified KRAS variants, including p.G12D, in small numbers (<1%) of WT, although most tumors contain multiple genetic aberrations.^18,19 Pathogenic FBXW7 variants were identified in up to 4% of sporadic WT ²⁰ and in rare cases of familial WT. ²¹ Our identification of a secondary cancer hotspot mutation in FBXW7 (p.R479G) in two of the WT nodules but not in an associated NR suggests a role in the progression from NR to WT, raising the hypothesis that WTs associated with somatic mosaic overgrowth disorders require additional driver mutations. The lack of a second hit in the other morphologically indistinguishable nodules underscores the difficulties in reliably differentiating hyperplastic/adenomatous NR and WT in post-chemotherapy specimens.

In conclusion, we describe a case of bilateral NRs and WT and a unique unilateral complex vascular anomaly that resembles FAVA in a patient with a somatic RASopathy due to an underlying KRAS p.G12D mutation. Moreover, we document the first occurrence of a secondary somatic cancer hotspot mutation (FBXW7 p.R479G) in the WT but not in the associated NRs, suggesting that Wilms tumorigenesis in somatic overgrowth disorders may involve additional driver mutations.