The UMBRELLA SIOPRTSG 2016 Wilms tumour pathology and molecular biology protocol

 [fig_ref] Figure 1: Handling of specimen by pathologist A [/fig_ref] [bib_ref] The pathology of Wilms' tumour (nephroblastoma): the International Society of Paediatric Oncology..., Vujanic [/bib_ref] 

## 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). [bib_ref] Clinical impact of histologic subtypes in localized non-anaplastic nephroblastoma treated according to..., Weirich [/bib_ref]. 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).
10. Fix the specimen in formalin (24-48 hours).
11. Samples for histological examination include at least one complete sampled longitudinal slice of tumour and kidney surface, accompanied by a guide block [fig_ref] Figure 1: Handling of specimen by pathologist A [/fig_ref]. If available, use mega-cassettes as it makes histological assessment easier.
In addition, sample the following:
[formula] a) [/formula]
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 [bib_ref] Clinical impact of histologic subtypes in localized non-anaplastic nephroblastoma treated according to..., Weirich [/bib_ref]. 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.
[fig] Figure 1: Handling of specimen by pathologist A: Fresh tumour on arrival at the pathology department B: Bi-valved tumour after inking of the surface reveals a multifocal, partly cystic and haemorrhagic tumour that replaces most of the normal kidney parenchyma C and D: examples of a guide block to indicate where each tumour block is sampled Supplementary Box 1. HANDLING OF SPECIMENS BY THE PATHOLOGIST [/fig]
[table] 3: The low risk tumours are cystic partially differentiated nephroblastoma (treated by surgery only) and completely necrotic Wilms tumour. The five histological types of Wilms tumours included in the intermediate risk group are regressive type, epithelial type, stromal type, mixed type and focal anaplasia. High risk Wilms tumours are blastemal type and diffuse anaplasia type. All Wilms tumour types are shortly described in the following section. In order to sub-classify and stage Wilms tumour tumour, it is essential to sample tumour according to the UMBRELLA Study protocol, and examine an adequate number of slides. If only a few slides are available, the pathologist should only make the primary diagnosis but should not try to sub-classify tumour.The diagnosis of a completely necrotic Wilms tumour is made when the tumour shows only regressive and/or necrotic changes caused by chemotherapy, in the absence of any viable tumour tissue, especially nests of blastema. The presence of scattered mature tubules, stroma and very small groups of blastemal cells is allowed, as they may represent remnants of nephrogenic rests and is not regarded as viable tumour. The typical histological appearance of pre-treated Wilms tumours is a mixture of necrosis, fibrosis, fibromyxomatous stroma containing lipid-and/or hemosiderin-laden macrophages. Necrosis is most often of coagulative-type. To identify 'ghost' structures a reticulin stain may be useful. Although complete tumour necrosis makes histological diagnosis of any tumour impossible, in many cases 'ghost' tumour structures (mainly blastema, occasionally epithelial elements) can be recognised. In addition, the presence of nephrogenic rests is a very reliable clue that the tumour was a Wilms tumour. Finally, if treated with pre-operative chemotherapy for Wilms tumour, other renal tumours (such as clear cell sarcoma of the kidney, rhabdoid tumour of the kidney, renal cell carcinoma) show only minimal to moderate chemotherapy-induced changes 4 . [/table]
[table] TABLE 2: The rest of the tumour can be stromal and blastemal, but only up to 10% of [/table]