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Anaplastic sarcoma of the kidney was first described as a novel pediatric renal neoplasm in 2007.1 Previous to this formal description of anaplastic sarcoma of the kidney, some of these lesions could have been included in renal tumors broadly characterized as embryonal sarcomas of the kidney.2, 3 In a series of 25 such tumors,2 3 possessed anaplasia, so perhaps these tumors could be considered to be unrecognized anaplastic sarcomas of the kidney. Others may have been primary renal synovial sarcomas.4 The one almost certain anaplastic sarcoma of the kidney identified before the paper of Vujanic et al in 2007 was published by Faria and Zerbini5 as a dedifferentiated cystic nephroma. In this case, the renal tumor occurred in a 26-month-old girl. It was largely cystic, but had a solid anaplastic region with cartilaginous and rhabdomyoblastic differentiation.5 In view of subsequent findings, it is perhaps surprising that neither germline nor somatic DICER1 mutations were identified in this person or the tumor 18 years later.6

Anaplastic sarcoma of the kidney presents as a large renal mass, and its major gross and histologic features include the presence of cysts, marked anaplasia in the spindle cell component, and areas of benign or malignant cartilage or chondroid differentiation.1 There is female predominance. In 2007, there were no definitive genetic mutations or molecular markers linked to these tumors. These tumors remain amongst the rarest renal neoplasms, with no more than 25 cases reported to date.

Recent studies have reported that a few isolated cases of ASK harbor mutations in DICER1,6, 7, 8, 9 making a case that anaplastic sarcomas of the kidney should be included with the other lesions of the pleiotropic tumor predisposition syndrome known as DICER1 syndrome (OMIM 606241). DICER1 syndrome tumors include pleuropulmonary blastoma,10 cystic nephroma,11 and many other rare tumor entities, mainly occurring in the pediatric and adolescent age range.12 DICER1 is an endoribonuclease central to generating microRNAs (miRNAs), small RNA molecules that downregulate the expression of ~30% of protein-coding genes.12 DICER1 utilizes its RNase IIIa and IIIb endonuclease domains to cleave precursor (pre)-miRNA stemloops, thus releasing the mature single-strand miRNA. Of note, mature miRNAs can be coded within either the 5′ (5p) or 3′ (3p) arms of pre-miRNA stemloops.12 DICER1-related tumors usually possess two DICER1 mutations: one predicted to result in a truncated protein and the other a missense mutation at specific residues within exons encoding the RNase IIIb domain of the DICER1 protein.12 Previous studies of DICER1 syndrome-related tumors have shown that when two DICER1 mutations are present in the tumor, one mutation is present on one of the two DICER1 alleles and the other is present on the alternate allele13, 14, 15, 16, 17, 18, 19, 20 (ie, the mutations are said to be in trans, or are biallelic). In contrast, if both the mutations occur on one allele, then they are in cis, or monoallelic.

Pleuropulmonary blastomas with a germline DICER1 mutation can occur with and without deleterious mutation in TP53.13, 18 Pleuropulmonary blastoma and anaplastic sarcoma of the kidney could be analogous tumors occurring in different organs; it has been suggested that the stages of DICER1-dependent pleuropulmonary blastomas could be reminiscent of the possible progression of DICER1-dependent cystic nephroma to anaplastic sarcoma of the kidney6 but it is unknown whether all anaplastic sarcomas of the kidney arise from pre-existing cystic nephromas,1, 5, 6, 7, 8, 9, 21, 22, 23 partly because of the rarity of anaplastic sarcomas of the kidney but also because the surgical removal of cystic nephromas will prevent the observation of development of anaplastic sarcoma of the kidney from cystic nephroma. Nevertheless, cystic nephromas can pre-exist in regions of the kidney where anaplastic sarcoma of the kidney have later been observed.6, 7, 8 In this report, we sought to determine in nine anaplastic sarcomas of the kidney—(1) the frequency of DICER1 mutations; (2) whether these mutations are biallelic; (3) whether the identified DICER1 mutations occurring in the RNase IIIb domain affect pre-miRNA processing; and (4) does aberrant TP53 status always accompany DICER1 mutations in anaplastic sarcomas of the kidney.

Materials and methods

Sample Acquisition and Histopathological Description of the Anaplastic Sarcomas of the Kidney

Nine anaplastic sarcomas of the kidney and matched normal kidney formalin-fixed paraffin-embedded specimens samples were obtained. Cases 1, 2, and 6 were described in the original description of anaplastic sarcoma of the kidney1 and case 8 was presented by current author Watanabe,23 however neither study investigated DICER1 mutation status. Cases 7 and 9 have been reported by our group (refs 7 and 8, respectively) as DICER1-related anaplastic sarcomas of the kidney. The diagnosis in case 5 was more equivocal than for the other eight cases, and it was considered that it could represent an anaplastic Wilms tumor (the original diagnosis, Table 1).

Table 1 Clinicopathological features and follow-up
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The study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine of McGill University, Montreal, QC, Canada (numbers A05-M60-14B, A08-M61-09B, and A12-M117-11A). Participants were recruited to the study in compliance with the second edition of the Canadian Tri-Council Policy Statement of Ethical Conduct of Research involving Humans and, where indicated because of young participant age, eligible relatives signed a consent form in accordance with the above-mentioned IRB protocols.

The anaplastic sarcomas of the kidney showed characteristic gross and histological features, including the presence of cysts (in the majority of cases) and solid areas composed of undifferentiated spindle cells with marked anaplastic changes, and prominent areas of benign or malignant cartilage or chondroid differentiation.1 Other, less common features include blastema-like areas, foci of rhabdomyoblastic differentiation, and small islands of osteoid.1

Screening DICER1 Mutations in Anaplastic Sarcoma of the Kidney

DNA was extracted from formalin-fixed paraffin-embedded samples and DICER1 RNase IIIa/b domain Sanger sequencing was performed as previously described.20 The ability of missense mutations to cause exon skipping was assessed as reported by our group.20 Additional fresh frozen tissue was available for three samples. In these cases, full DICER1 Sanger sequencing was performed and the phase of mutations was determined via cloning20 (Supplementary Data). Phase refers to whether two mutations are on the same copy of the gene (in cis) or whether there is one mutation on each chromosomal copy of DICER1 (in trans or biallelic). In addition, DNA extracted from formalin-fixed paraffin-embedded tissues (normal and tumor) was subjected to a custom-designed standard HaloPlex panel containing 11 genes (full gene or targeted regions of DICER1, SMARCA4, SMARCB2, CTNNB1, APC, BRAF, and PTCH1 as well as the exonic regions of DROSHA, FGF3, FGFR1, and TP53) according to a modified version of a previously published protocol.24 This gene panel was designed by our group to use for several projects; however, only DICER1 and TP53 were of interest for the current study. The subsequent deep sequencing was performed at the McGill University and Genome Quebec Innovation Center. Sequence analysis was carried out using a modification of established protocols (Supplementary Materials).

In Vitro Cleavage Assay

HEK 293 cells were transduced to stably express FLAG-tagged versions of the somatically acquired DICER1 RNase IIIb mutations. The ability of the FLAG-immunoprecipitated mutant proteins to cleave internally radiolabeled in vitro-transcribed pre-miR122 was evaluated in a time course by autoradiography of RNA products resolved by denaturing urea polyacrylamide electrophoresis. The details of this assay have previously been described.19

Immunohistochemistry

Immunohistochemistry for cases 1–8 was performed at the Segal Cancer Centre Research Pathology Facility (Jewish General Hospital). Tissue samples were cut at 4 μm, placed on SuperFrost/Plus slides (Fisher), and dried overnight at 37 °C, before immunohistochemical processing. The slides were then loaded onto the Discovery XT Autostainer (Ventana Medical System). All solutions used for automated immunohistochemisty were from Ventana Medical System unless otherwise specified. Slides underwent de-paraffinization and heat-induced epitope retrieval (CC1 pre-diluted solution Ref: 950–124, standard protocol). Immunostaining for TP53 was performed in an automated manner using a heat protocol. Briefly, pre-diluted mouse monoclonal anti-TP53 antibody (Clone Bp53-11, Ventana Medical Systems) was applied for 32 min at 37 °C then followed by the appropriate detection kit (OmniMap anti-Mouse-HRP, Ref: 760–4310) for 8 min, followed by ChromoMap-DAB (Ref: 760–159). A negative control was performed by omission of the primary antibody. Slides were counterstained with hematoxylin. Sections were scanned using the Aperio A Turbo and analyzed by Drs G Vujanic and A Spatz. Case 9 was stained with Cell Marque antibody P53 (DO7) at 1:300 dilution.

Results

Histological characteristics of some of the anaplastic sarcomas of the kidney are described in the legend of Figure 1. Clinicopathological features and follow-up are presented in Table 1. Somatically acquired DICER1 RNase IIIb mutations were identified in eight of the nine anaplastic sarcomas of the kidney studied (Figure 2a). These were determined to be somatically acquired as the mutation is present in the tumor DNA but absent in the matched normal tissue. Seven manifested as missense mutations (cases 2: c.5437G>A [p.E1813K]; 3: c.5113G>A [p.E1705K]; 4 and 7: c.5425G>A [p.G1809R]; 6 and 8: c.5125G>A [p.D1709N]; and 8 (second hit): c.5138A>T [p.D1713V]; Figure 2). In cases 1 and 9 the identified variant c.5438A>G has the potential to cause exon skipping (p.E1788fsX41) in addition to resulting in an altered amino acid (p.E1813G).8 Inactivating DICER1 mutations were observed in cases 1, 6, 7, and 9 (c.5023_5025delTACinsAG [p.Y1675RfsX30]; c.2026C>T [p.R676X]; c.2062C>T [p.R688X]; and c.2450delC [p.P817LfsX15], respectively; Figure 2a). As these inactivating mutations were also observed in matched adjacent normal tissue, we deemed them to be of germline origin but cannot exclude the possibility of a mosaic origin (Figure 2a). Inactivating mutations were also detected in cases 2 (c.4684_4685inC [p.C1562SfsX34]) and 3 (c. 1630C>T [p.R544X]; Figure 2a). The inactivating mutations in these two cases are likely somatically acquired as they were not present in matched normal tissue (Figure 2a; Supplementary Table 2). Cloning experiments to determine the phase of mutations in cases 7, 8, and 9 determined that the pairs of DICER1 mutations in each cases were most likely biallelic for cases 7 and 8, and show a trend toward biallelism in case 9 (see comment in Supplementary Data). The DICER1 mutations in cases 1, 2, and 3 are also presumed to be biallelic based on the DICER1 mutation phase analyses of other DICER1 syndrome lesions13, 14, 15, 16, 17, 18, 19, 20 but we were unable to confirm this presumption due to lack of fresh frozen tissue on which to perform cloning experiments. In summary, 8/9 anaplastic sarcomas of the kidney possessed at least one DICER1 mutation and 7/8 contained two DICER1 mutations (likely in trans, see above). In 3 of these 7 cases, both mutations seen in the tumor were of somatic origin (cases 2, 3, and 8; Figure 2b). In 4 of the 7 cases where at least one DICER1 mutation was present, the truncating mutation was also detected in adjacent normal tissue, and therefore was likely to be either mosaic or germline in nature (cases 1, 6, 7, and 9; Figure 2b; Supplementary Table 2).

Figure 1
figure 1figure 1

Anaplastic sarcoma of the kidney. (a) Small cysts surrounded by the stromal component, showing marked pleomorphism in (b); (c) anaplastic changes in the stroma and two islands of cartilage, one (upper) showing anaplastic changes; (d) an island of cartilage in the blastema-like area; (e) Case 8 H&E stained section at × 10 shows multifocal islands of cartilage and small cysts in the blastema-like area with necrosis. (f) Case 9 H&E stained section at × 200 shows a predominantly round and ovoid cell population expanding the wall of a cystic structure, which is lined by hobnail epithelium (black arrow). Marked nuclear pleomorphism features in this anaplastic sarcoma (white arrows). (g–j) Case 5. (g) Areas showing prominent rhabdomyoblastic differentiation. (h) A similar area with rhabdomyoblastic differentiation; (i) a spindle cell component of the tumor with some pleomorphic cells; (j) a mixture of areas with spindle cell and rhabdomyoblastic differentiation.

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Figure 2
figure 2

DICER1 mutations in anaplastic sarcomas of the kidney. (a) Table showing the DICER1 RNase IIIb mutations identified via Sanger sequencing of the hotspots or HaloPlex accompanying Sanger sequence verification. ‘Undetected’=no mutation was detected by HaloPlex; ‘n/a’=DICER1 exons were not analyzed as no DICER1 RNase IIIb mutations were identified in the case 5; ‘none’=no mutation was found by Sanger sequencing (RNAse IIIb domain) or despite adequate sequencing coverage (HaloPlex for remaining DICER1 exons); ‘failed’=PCR failed. (b) Position of mutations identified in the cases as represented as protein changes on a linear cartoon of the DICER1 protein. *This case has been previously reported.7 **This case has been previously reported.8 The DICER1 mutations in cases 7 and 8 were confirmed to be biallelic by cloning (Supplementary Data). ¢=this mutation was detected by HaloPlex in both normal and anaplastic sarcoma of the kidney tissues and we suggest that it is a germline mutation (Supplementary Table 2).

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The in vitro cleavage data demonstrate that all the DICER1 RNase IIIb mutations, when acting as missense mutations, are incapable of producing 5p miRNAs but instead produce 3p and an incompletely processed RNA molecule we term 5p+loop (Figure 3). If the DICER1 RNAse IIIb mutation of cases 1 and 9 produce a protein lacking exon 25, then no miRNAs are produced.8

Figure 3
figure 3

In vitro cleavage assay demonstrates that all DICER1 RNase IIIb mutations affect the production of 5p miRNAs. Using pre-miR122 as a surrogate for all DICER1-dependent pre-miRNAs, either the presence of 5p+loop and/or absence of 5p could be responsible for altering the transcriptome to initiate aberrant cellular signaling cascades to result in anaplastic sarcoma of the kidney. The somatic mutation in case 4 (c.5425G>A) is the same as that was previously reported for case 7, which results in 5p+loop and 3p.7 The somatic mutation (c.5425G>A) in cases 6 and 8 has been evaluated and results in 5p+loop and 3p. The somatic mutation in case 1 (c.5438A>G) is the same as that was reported for case 9, which can either result in 5p+loop and 3p (if it behaves as a missense mutation) or no miRNA generation (if it results in exon skipping).8

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The co-occurrence of a TP53 mutation with DICER1 was confirmed in three out of nine cases: cases 1 (c.630G>C [p.R210S]); 4 (c.212G>T [p.R71L]); and 6 (c.521G>A [p.R174Q]). Six of nine anaplastic sarcomas of the kidney (namely, cases 1, 4, 5, 6, 8, and 9) had aberrant TP53 immunohistochemistry (Figure 4a).

Figure 4
figure 4figure 4

TP53 status is altered in a subset of anaplastic sarcomas of the kidney. (a) Immunohistochemistry staining. Case 1 shows strong positivity in ~50% of tumor cells; case 2 is negative; case 3 is negative; case 4 shows strong positivity in 75–80% of tumor cells; case 5 shows strong positivity in ~30% of tumor cells; case 6—strong positivity in 25–30% of tumor cells; case 7—at × 40 magnification, negative; case 8—moderate positivity in ~85% of tumor cells; case 9—over 90% of cells show very strong positivity. Scale bar is shown for cases 1–6, 8, and 9. (b) Damaging TP53 mutations were identified either by HaloPlex and/or Sanger sequencing.

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Discussion

To our knowledge, this is the largest collection of anaplastic sarcomas of the kidney from which DICER1 sequencing has been attempted. There is a high prevalence of somatically acquired DICER1 RNase IIIb mutations in our collection (8/9; Figure 2), which we suggest is an important genetic feature of the disease. Biallelic DICER1 mutations were observed in two anaplastic sarcomas of the kidney (cases 7 and 8) and a trend toward biallelism was seen in the third (case 9) where this could be assessed. In the case of cases 2, 3, and 8, the DICER1 mutations were both somatically acquired (Figure 2a). Therefore, the two-hit hypothesis of tumor formation is supported by the data for DICER1 mutations.

All the somatically acquired DICER1 RNase IIIb mutations seen in these anaplastic sarcomas of the kidney affect the cleavage of the 5p arm of the pre-miR122 stemloop (Figure 3). Using pre-miR122 as a surrogate for all DICER1-dependent pre-miRNAs, it appears that the tumors with the combination of one inactivating and one RNase IIIb DICER1 mutations are deficient in producing 5p miRNAs and/or have deleterious effects due to the presence of 5p plus loop RNA structures as we and others have suggested in previous studies.7, 8, 19, 25 Alternatively, the presence of only 3p miRNAs could predispose a cell to tumorigenesis. Given the observation that inherited mutations in genes involved in miRNA processing have been observed in patients with Wilms tumor20, 26 and cystic nephroma,27 it has been suggested that availability of a wide range of miRNAs is important for kidney development.26 It seems clear that pediatric cystic nephroma27 and much more rarely, Wilms tumor20, 26, 28, 29 can arise from the kidney of a proband with an inherited DICER1-inactivating mutation, but that the pathway to Wilms tumor does not appear to pass through a pre-existing pediatric cystic nephroma (Figure 5). On the other hand, our recent case report of a microscopic nascent anaplastic sarcoma of the kidney occurring in a pediatric cystic nephroma8 (case 9 here), taken together with previous publications,6, 7 further supports the notion that an anaplastic sarcoma of the kidney can arise within a cystic nephroma (Figure 5).

Figure 5
figure 5

Model depicting possible evolution of DICER1-affected kidney tumors. Dotted arrows represent rare events, solid lines represent more common events, arrows with dots and dashes represent events with unknown frequencies. Wilms tumor with biallelic DICER1 mutations is rarely observed,20 but it seems likely that many anaplastic sarcomas of the kidney arise in pre-existing pediatric cystic nephromas.6, 7, 8

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Overexpressed TP53 as observed by immunohistochemistry as seen in 6/9 of the anaplastic sarcomas of the kidney (Figure 4a) has been used as a surrogate method of TP53 mutation status. In addition, potentially damaging TP53 mutations were identified by HaloPlex in cases 1, 4, and 6 (Figure 4b; Supplementary Table 3). We suggest that mutations in the promoter, 5′-UTR, or 3′-UTR of TP53 not included in the Sanger sequencing and HaloPlex could account for lack of mutations detected in cases 5, 8, and 9. Nevertheless, our reported proportion of anaplastic sarcomas of the kidney with aberrant TP53 expression by immunohistochemistry is similar with that reported for DICER1-dependent pleuropulmonary blastomas.18

Case 5, which showed neither DICER1 nor TP53 mutations, but did overexpress TP53, shared some diagnostic features of anaplastic sarcomas of the kidney (Figures 1g–j), such as widespread anaplastic changes, but lacked some other commonly seen features such as cysts and convincing chondroid differentiation, making this case somewhat atypical and indistinguishable from anaplastic Wilms tumor. In addition, as this was the only tumor that we studied here that did not possess a DICER1 mutation, and as DICER1 mutations are much rarer in Wilms tumors than in anaplastic sarcomas of the kidney, we are inclined, in retrospect, to consider this to be an anaplastic Wilms tumor.

This study suggests that identification of DICER1 RNase IIIb mutations is a useful genetic marker for anaplastic sarcomas of the kidney. Also, DICER1-dependent evolution from cystic nephroma to anaplastic sarcoma of the kidney may parallel the evolution of DICER1-dependent type I cystic pleuropulmonary blastomas to more solid types II and III pleuropulmonary blastomas. As aberrant immunohistochemistry status of TP53 was only seen in a proportion of anaplastic sarcomas of the kidney, it is not a useful molecular marker of the disease. Given the high incidence of DICER1 mutations in our set of anaplastic sarcomas of the kidney we suggest that screening for both germline and somatic DICER1 mutations is warranted in suspected cases of anaplastic sarcoma of the kidney.