The UMBRELLA SIOP–RTSG 2016 Wilms tumour pathology and molecular biology protocol

On the basis of the results of previous national and international trials and studies, the Renal Tumour Study Group of the International Society of Paediatric Oncology (SIOP–RTSG) has developed a new study protocol for paediatric renal tumours: the UMBRELLA SIOP–RTSG 2016 protocol (the UMBRELLA protocol). Currently, the overall outcomes of patients with Wilms tumour are excellent, but subgroups with poor prognosis and increased relapse rates still exist. The identification of these subgroups is of utmost importance to improve treatment stratification, which might lead to reduction of the direct and late effects of chemotherapy. The UMBRELLA protocol aims to validate new prognostic factors, such as blastemal tumour volume and molecular markers, to further improve outcome. To achieve this aim, large, international, high-quality databases are needed, which dictate optimization and international harmonization of specimen handling and comprehensive sampling of biological material, refine definitions and improve logistics for expert review. To promote broad implementation of the UMBRELLA protocol, the updated SIOP–RTSG pathology and molecular biology protocol for Wilms tumours has been outlined, which is a consensus from the SIOP–RTSG pathology panel.

Intact surgical specimens are delivered fresh to the pathology department without being incised by the surgeon. Preferably, the surgeon marks important areas that need attention, and accompanies the specimen to the pathology department to discuss the specimen with the pathologist directly. As soon as it arrives at the pathology department, processing starts in order to minimize degradation of DNA and RNA. The examination of the specimen includes: 1. Weighing, measuring and photographing the whole specimen (FIG. S1A). Look carefully for ruptures/fissures and ink them in a different colour then the rest of the specimen.
2. Search for and dissect the peri-renal and peri-hilar lymph nodes. Record the presence of (suspicion of) tumour and/or necrosis. Block these separately, recording their sites.
3. Identify the renal vein, artery and ureter and take transverse sections of each at/near the resection margin. 4. Ink the surface of the whole specimen and renal sinus with Indian ink. This is a critical step and always needs to be done, otherwise it might be impossible to stage the tumour accurately.

5.
Open the specimen by a longitudinal incision (bi-valve) to demonstrate the tumour and its relation to the kidney, capsule, and renal sinus (FIG. S1B). 6. Photograph the tumour, and record the macroscopic appearance.
7. Measure the tumour in all three dimensions, since this will be used for calculating volume.
8. Assess the percentage of necrotic tumour.
9. Take samples required for biology studies (described in the main text).
11. Samples for histological examination include at least one complete sampled longitudinal slice of tumour and kidney surface, accompanied by a guide block (FIG. S1C and S1D). If available, use mega-cassettes as it makes histological assessment easier.
In addition, sample the following: Areas of the tumour that look different macroscopically. It is strongly recommended to state all relevant histological findings with its corresponding block/slide number (for example, 'renal sinus invasion in block A7') as it makes central pathology review easier. It is recommended to make two sets of all sampled blocks at the same time, and send the second set for rapid central pathology review, even if only with a provisional report (final report can be emailed once it is ready).

Supplementary Box 2. HISTOLOGICAL CLASSIFICATION
Beckwith and Palmer's criteria for histological sub-typing of Wilms tumours state that one component has to comprise at least 2/3 (66%) of a tumour mass for the tumour to be sub-classified accordingly 1 .
Pre-operative chemotherapy results in so-called 'chemotherapy-induced changes' in many Wilms tumours. Therefore, the criteria applicable to sub-classification of primarily operated tumours are modified to take these changes into account and distinguish three different prognostic sub-groups: low, intermediate, and high risk 2,3 .

Epithelial type Wilms tumour
The criteria for diagnosing epithelial type Wilms tumour are that the viable part of the tumour must comprise more than 1/3 of the tumour, and of the viable tumour, at least 2/3 consists of epithelial structures ( Stromal type Wilms tumour usually occurs in younger children 5 . However, stromal differentiation may be induced by pre-operative chemotherapy as a stromal type Wilms tumour is far more common in children who have received pre-operative chemotherapy 6 . Stromal type Wilms tumours usually show minimal to moderate chemotherapy-induced changes, since stromal tissue is usually resistant to chemotherapy 5,6 .

Mixed type Wilms tumour
The histological criteria for making a diagnosis of mixed type nephroblastoma are: a) the viable part of the tumour comprises more than 1/3 of a tumour mass; b) the viable tumour consists of blastemal and/or epithelial and/or stromal elements, but none of them comprise more than 2/3 of the viable tumour; c) tumours which contain >10% of blastema, even if the predominant components are epithelial or stromal components.

Regressive type Wilms tumour
In regressive type Wilms tumour, the previously described chemotherapy-induced changes comprise more than 2/3 of the tumour mass, irrespectively of what the viable part of tumour is (except for diffuse anaplasia). The assessment of the percentage of necrosis/regression is done on both gross and histological examination. Therefore, blocks are taken not only from viable parts of the tumour mass, but also from the parts that show necrotic/regressive changes.

Wilms tumour with focal anaplasia
Wilms tumours which contain one or two foci of anaplasia according to the established criteria (see main text), are still sub-typed and should be reported as, for example, 'Wilms tumour, mixed type with focal anaplasia'. Generally, the size of an anaplastic focus should not exceed 15 mm. However, it is still important to determine the underling sub-type. If the underlying sub-type is blastemal, it is classified as a high risk tumour.

Blastemal type Wilms tumour
In blastemal type Wilms tumour, the viable part of the pre-treated tumour must comprise more than 1/3 of the tumour mass, and at least 2/3 of the viable tumour consists of blastema. The blastemal volume is discussed separately as a new item of the pathology protocol in the main text. The blastemal elements are composed of undifferentiated round or elongated cells, which are usually closely packed, and show no evidence of epithelial and/or stromal differentiation. There are several distinctive patterns (e.g. diffuse, serpentine) in which blastemal cells may occur, but they are of no prognostic or therapeutic significance.

Wilms tumour with diffuse anaplasia
Anaplasia occurs in about 5-8% of patients with Wilms tumour 1 . The finding of diffuse anaplasia overrules the diagnosis of any other sub-type of Wilms tumour (for example, it may occur in regressive Wilms tumour), and always upgrades the Wilms tumour to the high risk category. The Wilms tumour with diffuse anaplasia as well as the distinction of focal versus diffuse anaplasia are described in the main article text.