Long-term kidney function in children with Wilms tumour and constitutional WT1 pathogenic variant

We identified retrospectively from hospital records and the oncology departmental database of patients with kidney tumours, cases of patients with co-existing WT and constitutional WT1 pathogenic variant who received treatment between 1993 and 2016 at Great Ormond Street Hospital for Children NHS Foundation Trust (GOSH), UK. WT1 pathogenic variant testing was done at different points for each patient as directed by the clinical presentation. This could be at diagnosis, during treatment, or at follow-up particularly in the older cases. Patients with WAGR and DDS were included. WAGR patients were defined as those with the full complement of WT, aniridia (AN), GU malformations, and intellectual disability (MIM#194072). DDS patients were defined as having WT, nephropathy presenting as persistent proteinuria or overt nephrotic syndrome, and GU anomalies (MIM#194080) [17, 18]. WT patients with only some of these phenotype anomalies were categorized by their specific features (AN, diffuse mesangial sclerosis (DMS), and GU malformations).

We collected data on patient and tumour demographics which comprised patient gender, age at diagnosis, and phenotypic anomalies; tumour histology, laterality, multifocality, tumour volume at diagnosis and after pre-operative treatment; and treatment details of chemotherapy, type of surgery, and radiation received.

All patients had pre-operative chemotherapy and tumour histological subtype was according to the revised SIOP classification [19]. For tumours classified initially according to other protocols, a second pathological review was performed by an expert pediatric pathologist (WM) for the purposes of this study. Tumours were also categorized according to the presence or absence of rhabdomyoblastic differentiation and intralobar and perilobar nephrogenic rests (ILNR and PLNR, respectively).

All WT1 pathogenic variants and deletions were identified from peripheral blood lymphocytes by an NHS genetic diagnostic service, using Sanger sequencing, MLPA (multiplex ligation-dependent probe amplification), and, in older cases, FISH (fluorescent in situ hybridization). Non-syndromic patients with WT in our service were selected for constitutional WT1 pathogenic variant testing at the discretion of the treating clinician based on previously published criteria suggesting a higher risk of such pathogenic variant [13] or when they had some features of DDS in the absence of urogenital malformation (persistent hypertension or proteinuria).

We assessed the following data at diagnosis and at the last follow-up: kidney function with serum creatinine and estimated glomerular filtration rate (eGFR), urine albumin to creatinine ratio, blood pressure, and use of anti-hypertensiveand/oranti-proteinuric medications.

The GFR was estimated using the revised Schwartz formula: eGFR (ml/min/1.73 m²) = k × height (cm)/serum creatinine (μmol/l) with a k value of 33 [20, 21]. If the height of the patient was missing, it was estimated from the percentile growth chart of the patient. The eGFR was used to stage the kidney function of the patients according to standard criteria [22]. Albuminuria was defined as a urine albumin to creatinine ratio above the age-adjusted normal range.

WT patients were categorized as having kidney failure if they received chronic kidney replacement therapy with dialysis or transplantation. The malignancy was regarded as the cause of kidney failure if the patient needed surgical removal of all the kidney tissue as a result of widespread/progressive bilateral WT or a relapse in the solitary kidney that required nephrectomy.

Numerical variables were summarized as medians and standard deviation as the data followed a non-parametric distribution. Categorical and ordinal variables were described as relative frequencies. Medians and frequencies were compared using Mann-Whitney’s U test and the Kruskal-Wallis test respectively. All tests were two-sided and a p value = 0.05 was considered significant. All statistical analysis was performed using SPSS© (version 24.0; SPSS Inc. Chicago, IL).

We identified 26 patients with WT and constitutional WT1 pathogenic variant and one patient with a complete phenotype of WAGR syndrome who had no documented analysis of WT1 pathogenic variant but is assumed to have a constitutional deletion (Table 1). We excluded two patients, one with less than 6 months of follow-up from the date of diagnosis and one without available data on kidney function. Fifteen were male and median age at diagnosis of the whole cohort was 14 months (range 4–74 months).

Fifteen (60%) patients (13 males, 2 females) had associated phenotypic anomalies: WAGR (n = 2; 8%); AN (n = 1; 4%); incomplete DDS (n = 1; 4%); GU malformations (n = 11; 44%) including cryptorchidism, hypospadias, and micropenis. In the remaining 10 (40%) patients, no phenotypic anomalies were found. In particular, no GU abnormalities were reported in any female patients (Table 1). One unusual male patient carried the splice site pathogenic variant associated with Frasier syndrome that prevents formation of the +KTS isoform of WT1 but lacked any GU malformation. He presented at an older age with WT (74 months) and had albuminuria (albumin to creatinine ratio 140 mg/mmol) but normal creatinine and GFR. His kidney function deteriorated slowly over the subsequent 7 years at which time transplant was recommended. Another female patient had unilateral WT without other abnormal clinical features but presented with nephrotic syndrome 3 years after her WT nephrectomy. She developed stage 5 chronic kidney disease 5 years later, underwent second nephrectomy with evidence of nephroblastomatosis, and received a transplant 11 years later.

The patients were subdivided according to type of WT1 pathogenic variant into the following subgroups (Table 2): large WAGR deletions encompassing the 11p13 region and entire WT1 gene (4 patients; note one deletion did not extend into the PAX6 gene); missense (2 patients); nonsense (8 patients); frameshift (7 patients); and splice site pathogenic variant (4 patients). The patient with full WAGR phenotype but no genetic testing is assumed to carry deletion of the 11p13 region.

The median age at WT diagnosis according to pathogenic variant type was 9 months (3.8–62.9) frameshift, 13 months (7.5–74.3) splice site, 14 months (5.5–29.6) nonsense, 15 months (12.6–17.6) missense, and 24 months (14.0–47.5) deletions for their respective pathogenic variant types.

We observed variation in the frequency of bilateral tumours by pathogenic variant type, 7/8 patients carrying nonsense pathogenic variants compared with frameshift (3/7), splice site (2/4), deletions (1/4), and missense (0/2), but this did not reach statistical significance (p = 0.192; Tables 1 and 2). Both missense variants affect functionally important residues in zinc finger 3, base-contacting residue Arg394, and zinc co-ordination residue His405.

All patients had pre-operative chemotherapy with 18 patients treated according to the SIOP 2001 protocol and seven according to similar previous national protocols. Among the 13 patients with bilateral tumours, two underwent up-front bilateral nephrectomies, three had bilateral NSS, and eight unilateral nephrectomy with contralateral NSS. Of the 12 patients with unilateral tumour, one had bilateral nephrectomies due to kidney failure, two underwent unilateral NSS, and nine had unilateral nephrectomy of whom two subsequently had metachronous relapse treated by contralateral nephrectomy in one case and contralateral NSS in the other. Ultimately, 21/25 (84%) patients retained some functioning kidney tissue.

Five (20%) patients received radiotherapy, with four patients receiving unilateral flank radiotherapy due to abdominal stage III WT, and one patient whole lung radiotherapy due to pulmonary metastases.

Patient overall survival was 100%, with three patients in remission after disease relapse, and kidney survival was 72% at median follow-up of 9 years. Seven patients (28%) commenced chronic dialysis of which three were after bilateral nephrectomies (one patient had subsequent contralateral nephrectomy due to relapse). The median time between the diagnosis and the start of haemodialysis was 5.6 (0–16) years (Table 3). Kidney survival (time from diagnosis of WT to start dialysis) is shown by the Kaplan-Meier graph in Fig. 1. The overall kidney survival for this cohort as mean time to start of dialysis was 13.38 years (95% CI: 10.3–16.4), where 7 patients reached kidney failure at a median of 5.6 years. All of these seven patients were subsequently transplanted. Twelve patients had albuminuria and/or were prescribed ACE inhibitors. Two of these patients had albuminuria only, while among ten patients who were commenced on medication, six were being treated for hypertension and four were receiving anti-proteinuric medication. Two patients had stage III and stage IV chronic kidney disease (CKD). Four patients (3 frameshift; 1 WT1 deletion) had normal blood pressure and kidney function without proteinuria at follow-up from 1.5 to 12 years (Table 4).

In each individual patient, we assessed the frequency of the following clinical/histologic factors suggestive of potential WT1 pathogenic variant: age at diagnosis < 12 months, bilateral WT, GU malformation, ILNR, DMS, rhabdomyoblastic differentiation, albuminuria at diagnosis, and persistent hypertension after nephrectomy (Table 5). Nineteen of 25 patients had at least three or more of these clinical/histologic features, two patients had two characteristic features (GU malformation and ILNR), and the remaining four patients had only one feature, although two of these also had a WT1-associated syndrome.