
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01909-6
10.1016/j.isci.2024.110684
110684
Article
Widening the spectrum of players affected by genetic changes in Wilms tumor relapse
Ciceri Sara 1
Bertolotti Alessia 2
Serra Annalisa 3
Gattuso Giovanna 4
Boschetti Luna 4
Capasso Maria 5
Cecchi Cecilia 6
Sorrentino Stefania 7
Quarello Paola 89
Ciniselli Chiara Maura 10
Verderio Paolo 10
De Cecco Loris 11
Manenti Giacomo 12
Diomedi Camassei Francesca 13
Collini Paola 14
Spreafico Filippo 415
Perotti Daniela daniela.perotti@istitutotumori.mi.it
11516∗
1 Predictive Medicine: Molecular Bases of Genetic Risk, Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
2 Diagnostic and Molecular Research Lab, Department of Advanced Diagnostics, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
3 Department of Pediatric Hematology and Oncology, Gene and Cellular Therapy, Bambino Gesù Children’s Hospital IRCCS, Rome, Italy
4 Pediatric Oncology Unit, Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
5 Department of Pediatric Hemato-Oncology, AORN Santobono-Pausilipon, Naples, Italy
6 Division of Pediatric Oncology/Hematology, Meyer University Children’s Hospital, Florence, Italy
7 U.O.C. Oncologia Pediatrica, IRCCS Istituto Giannina Gaslini, Genova, Italy
8 Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, Regina Margherita Children’s Hospital, Turin, Italy
9 Department of Public Health and Pediatrics, University of Turin, Turin, Italy
10 Unit of Bioinformatics and Biostatistics, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
11 Integrated Biology of Rare Tumors, Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
12 Unit of Animal Health and Welfare, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
13 Pathology Unit, Department of Laboratories, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
14 Soft Tissue Tumor Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
∗ Corresponding author daniela.perotti@istitutotumori.mi.it
15 These authors contributed equally

16 Lead contact

06 8 2024
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© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Few studies investigated the genetics of relapsed Wilms tumor (WT), suggesting the SIX1 gene, the microRNA processing genes, and the MYCN network as possibly involved in a relevant percentage of relapses. We investigated 28 relapsing WT patients (10 new cases and 18 cases in which the involvement of SIX and miRNAPG had been excluded) with a panel of ∼5000 genes. We identified variants affecting genes involved in DNA damage prevention and repair in 12/28 relapsing patients (42.9%), and affecting genes involved in chromatin modification and regulation in 6/28 relapsing patients (21.4%), widening the spectrum of anomalies detected in relapsed tumors. The disclosure of molecular pathways possibly underlying tumor progression might allow to use molecularly targeted therapies at relapse. Surprisingly, germline anomalies, mostly affecting DNA damage prevention and repair genes, were identified in 13/28 patients (46.4%), raising the issue of performing a genetic testing to all children presenting with a WT.

Graphical abstract

Highlights

• We found variants in DNA damage prevention/repair genes in relapsing WT patients

• We found variants in chromatin modification/regulation genes in relapsing WT

• The best approach to a relapsed child is a personalized intervention

• Pathways underlying WT progression could facilitate molecular targeted therapies

Cancer; Clinical genetics; Genomics; Human genetics

Subject areas

Cancer
Clinical genetics
Genomics
Human genetics
Published: August 6, 2024
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pmcIntroduction

Wilms tumor (WT) represents a success in pediatric oncology, with approximately 90% of patients reaching a cure.1 However, for half of patients who experience a relapsing disease, WT remains mortal.1 The landscape of the genetics of primary WT has been well established,2,3 whereas the characterization of WT recurrence lagged behind, mainly due to the scarcity of material at relapse available for analyses. Up to date, four papers presented data on paired primary/relapsing WT specimens, with increasingly detailed description of the events underlying tumor progression. The first paper presented chromosomal data on 10 paired primary/relapsing tumors, disclosing gains at chromosomes 5p, 8p12, 15q, 16p, and 20q, and losses of 11q and 17p as events acquired in two recurrent tumors.4 We demonstrated, on 8 paired primary/relapsed tumors, chromosomal anomalies at 1q, 3, 16q in relapses, and in particular 1q gain and/or allelic imbalance in 3/8 (37.5%) primary tumor samples and in 6/8 (75%) paired relapse samples, suggesting the importance of this chromosomal anomaly at relapse.5 Targeted sequencing of SIX1 and SIX2 and of microRNA processing genes (miRNAPG) DICER1, DROSHA, and DGCR8 in 27 paired primary/relapsed WT revealed the co-occurrence of SIX1 and DROSHA mutations in the relapse of 4 patients in whom the primary tumors displayed the heterogeneous presence of DROSHA mutations, and the presence of a SIX1 mutation in only two cases.5,6 miRNAPG mutations were identified in 5 recurrences: as already observed, the presence of the mutation in the recurrence was disclosed in all the cells of all investigated samples, whereas the same mutation could be heterogeneously present (not in all the cells or the tumor blocks studied) in the primary disease.6 These evidences supported the role of SIX1 and miRNAPG anomalies in tumor progression.5,6 A recent paper on 51 favorable histology relapsed WT investigated by whole genomic sequencing, confirmed our previous observation on the role of chromosome 1q gain and SIX1 mutation acquisition in the progression of the tumor.7 Furthermore, the importance of the involvement of MYCN mutation and the MYCN pathway was disclosed, with 30% of relapses having a mutation affecting this pathway, and with two out of three MYCN P44L mutations being present only in the relapsing disease and not in the primary tumor, to suggest MYCN involvement in tumor progression.7 This study (like most performed on WT), relied on a single tumor sample, which could not be representative of the entire tumor mass, considering that intratumor genetic heterogeneity is a well-established phenomenon in WT.8 However, the analysis of the relapsed disease is expected to highlight the positive selection of the clonal events of the primary tumor involved in tumor progression and poor outcome.

What clearly emerges from all the genetic studies performed on WT is that primary WT typically arise after the acquisition of more than one genetic event and that many candidate driver genes are involved, such as WT1, CTNNB1, SIX1 and SIX2, DROSHA, DGCR8, MYCN, and AMER1. There remains, however, an important fraction of primary WT in which no mutation in such genes is found.2,3,7 Similarly, the scenario which is now emerging for WT relapses is heterogeneous, with multiple genes and pathways involved in tumor progression, and with the SIX1, miRNAPG, and MYCN pathway explaining only a fraction of relapses.5,6,7,9

In the present study, we investigated 28 cases of relapsed WT using the SOPHiA Clinical Exome Solution v2.0 panel, and through multiple samplings approach, we aimed to reconstructing the temporal acquisition of the identified variants.

Results

Genetic description of the investigated tumors

Following manual curation and Sanger validation, genetic variants in the panel of investigated genes were identified in 20/28 patients (71.4% of investigated cases) (Figure 1; Table 1; Table S1). Patient no. 3 displayed a TP53 splice region variant and a MUTYH nonsense variant in the primary tumor: both variants were in homo/hemizygosity in the primary tumor and in both recurrences, and furthermore, one recurrence acquired a BRCA2 missense variant. Patient no. 7 had two heterozygous variants in the primary disease: a PALB2 missense variant and the MYCN hotspot mutation P44L (for MYCN, sequencing was successful only in 1/3 samples), both these variants were also present in the second (and only available) recurrence, in which MYCN mutation was disclosed in all the samples in almost homo/hemizygosity. Patient no. 12 had two heterozygous missense variants in PALB2 and RNASEH2B in the primary tumor. No further variants were identified in the recurrence. Patient no. 14 displayed three heterozygous variants in the primary tumor: a BLM missense variant, a CHEK2 intronic variant affecting the splice polypyrimidine tract, and a CREBBP nonsense variant. The recurrence displayed these same variants, however the CREBBP variant, although still present, showed a reduced frequency of the variant allele (VAF). Patient no. 15 had a CTNNB1 missense variant occurring late during primary tumor development, since it was detected with a low VAF in only 1/3 fragments tested, in addition, also a LTBP3 missense variant occurred late during primary tumor development, as detected with a low VAF (sequencing failed in the 2 primary tumor blocks not investigated by next generation sequencing [NGS]); the first and the second recurrences displayed the CTNNB1 and LTBP3 missense variants, and furthermore a VCAN missense variant was acquired in the second recurrence; the third recurrence displayed only the CTNNB1 variant in common with the primary disease and acquired a missense variant in STIL. Patient no. 16 carried a PMS2 heterozygous missense variant both in primary and relapsing disease. Patient no. 18 displayed in the primary disease, in heterozygosity, a splice acceptor -2 variant of the TP53 gene, together with a PAX8 and a UBA1 frameshift variants. TP53 variant was reduced to homo/hemizygosity in 2/3 tissue fragments of primary disease. Both the metastatic lymphnode at diagnosis and the recurrent disease showed the same variants of the primary disease, with TP53 variant reduced to homo/hemizygosity. Patient no. 19 carried in the primary disease three heterozygous variants: a BRCA2 nonsense variant, a SMARCAL1 missense variant, and a SDHB splice donor +1 variant, together with a homozygous/hemizygous BMI1 splice acceptor -1 variant (found in homo/hemizygosity in all 3 available blocks). The three different relapses showed the same mutations of the primary disease. Patient no.28 displayed a heterozygous missense variant in DYNC1H1 in the primary disease (in 2/2 blocks) and in the recurrent disease. Patient no. 33 had three heterozygous variants in the primary disease: a BRCA2 missense variant, a CTC1 frameshift, and a DICER1 missense variants. No further anomalies were disclosed in the recurrence. Patient no. 34 displayed a BLM heterozygous missense variant both in primary and in the recurrent disease. Patient no. 35 had in the primary tumor two heterozygous variants affecting the CDC73 gene: a CDC73 splice acceptor -2 variant and a CDC73 no-start variant (affecting the starting methionine, present in all 3 blocks available), and furthermore, probably acquired later during primary tumor growth, the hotspot MYCN variant (present in 1/2 blocks tested); in two different recurrences a further OBSL1 missense variant was acquired. The subcutaneous mass in correspondence of the surgical scar carried only the two CDC73 variants. Patient no. 36 had a homo/hemizygous TP53 missense variant in all the three blocks of the primary tumor and acquired a CREBBP nonsense variant in the recurrence. Patient no. 36α had a heterozygous CTNNB1 missense variant occurring in the primary tumor, and no further anomalies acquired in the recurrent disease. Patient no. 39α carried in heterozygosity in the primary tumor two different variants in BLM, one missense and one nonsense, and furthermore, a FANCA homo/hemizygous missense variant and a KMT5B homo/hemizygous missense variant. The recurrence displayed the same mutations of the primary disease. Patient no. 111α had a heterozygous missense SMAD3 variant and a late occurring (with low VAF) TP53 missense variant in the primary tumor; in the recurring disease the TP53 VAF was closer to homo/hemizygosity and in addition the recurrent disease acquired a nonsense variant of the PRMT7 gene and a missense variant in the CHL1 gene. Patient no. 190 displayed heterozygous missense variants in the CHEK2 and in the DICER1 gene in primary disease; in the recurrence a nonsense variant in ATM was acquired and the CHEK2 variant presented in homo/hemizygosity. Patient no. 31 carried a BBS2 heterozygous frameshift variant in both recurrences; whereas a TUBGCP6 heterozygous nonsense variant was present in only one recurrence (primary tumor was not available). Patient no. 32 displayed in heterozygosity the co-occurrence of a frameshift and of a missense variant in the ATM gene in 1/2 samples of the first recurrence; no ATM variant was present in the second recurrence (primary tumor was not available). Patient no. 553 carried DIS3L2, ABCC2, KLF10, and ZFHX3 heterozygous missense variants in the recurrence (primary tumor was not available).Figure 1 Genetic anomalies detected in the panel of WT investigated

Genes: HUGO symbols; Genes in the red square: genes involved in DNA damage prevention and repair, genes in the blue square: genes involved in chromatin modification and regulation. NA: primary tumor not available; ∗: these data are expanded in the text, in Table 1 and in Table S1.

Table 1 Genetic anomalies detected in the panel of WT investigated

Patient	Germline	Primary tumor	Relapse	
3	TP53, splice region, het MUTYH, nonsense, het	I	TP53, splice region, H/H MUTYH, nonsense, H/H	IIa	TP53, splice region, H/H MUTYH, nonsense, H/H
BRCA2, missense, het	
IIb	TP53, splice region, H/H MUTYH, nonsense, H/H	
7	PALB2, missense, het	I	PALB2, missense, het MYCN, missense, het	III	PALB2, missense, het MYCN, missense, H/H	
12	PALB2, missense, het RNASEH2B, missense, het	I	PALB2, missense, het RNASEH2B, missense, het	II	PALB2, missense, het RNASEH2B, missense, het	
14	BLM, missense, het CHEK2, splice polypyr, het	I	BLM, missense, het CHEK2, splice polypyr, het CREBBP, nonsense, het	II	BLM, missense, het CHEK2, splice polypyr, het CREBBP, nonsense, het	
15		I	CTNNB1, missense, het LTBP3, missense, het	II	CTNNB1, missense, het LTBP3, missense, het	
III	CTNNB1, missense, het LTBP3, missense, het VCAN, missense, het	
IV	CTNNB1, missense, het STIL, missense, het	
16	PMS2, missense, het	I	PMS2, missense, het	II	PMS2, missense, het	
18		I	PAX8, frameshift, het TP53, splice acc −2, het-H/H UBA1, frameshift, het	II	PAX8, frameshift, het TP53, splice acc −2, H/H UBA1, frameshift, het	
Ln	PAX8, frameshift, het TP53, splice acc −2, H/H UBA1, frameshift, het	
19	BRCA2, nonsense, het SMARCAL1, missense, het SDHB, splice don +1, het	I	BRCA2, nonsense, het SMARCAL1, missense, het SDHB, splice don +1, het BMI1, splice acc −1, H/H	IIa	BRCA2, nonsense, het SMARCAL1, missense, het SDHB, splice don +1, het BMI1, splice acc −1, H/H	
IIb	BRCA2, nonsense, het SMARCAL1, missense, het SDHB, splice don +1, het BMI1, splice acc −1, H/H	
IIc	BRCA2, nonsense, het SMARCAL1, missense, het SDHB, splice don +1, het BMI1, splice acc −1, H/H	
28		I	DYNC1H1, missense, het	II	DYNC1H1, missense, het	
33	BRCA2, missense, het CTC1, frameshift, het	I	BRCA2, missense, het CTC1, frameshift, het DICER1, missense, het	III	BRCA2, missense, het CTC1, frameshift, het DICER1, missense, het	
34	BLM, missense, het	I	BLM, missense, het	III	BLM, missense, het	
35	CDC73, splice acc −2, het	I	CDC73, no start, het CDC73, splice acc −2, het MYCN, missense, het	IIs	CDC73, no start, het CDC73, splice acc −2, het	
IIa	CDC73, no start, het CDC73, splice acc −2, het MYCN, missense, het OBSL1, missense, het	
IIb	CDC73, no start, het CDC73, splice acc −2, het MYCN, missense, het OBSL1, missense, het	
36		I	TP53, missense, H/H	II	TP53, missense, het CREBBP, nonsense, het	
36α		I	CTNNB1, missense, het	II	CTNNB1, missense, het	
39α	BLM, missense, het BLM, nonsense, het	I	BLM, missense, het BLM, nonsense, het FANCA, missense, H/H KMT5B, missense, H/H	II	BLM, missense, het BLM, nonsense, het FANCA, missense, het KMT5B, missense, H/H	
111α		I	SMAD3, missense, het TP53, missense, het	II	SMAD3, missense, het TP53, missense, H/H PRMT7, nonsense, het CHL1, missense, het	
190	CHEK2, missense, het DICER1, missense, het	I	CHEK2, missense, het DICER1, missense, het	II	CHEK2, missense, H/H DICER1, missense, het ATM, nonsense, het	
31	BBS2, frameshift, het	I	NA	IIa	BBS2, frameshift, het	
IIb	BBS2, frameshift, het TUBGCP6, nonsense, het	
32		I	NA	II	ATM, frameshift, het ATM, missense, het	
III		
553	DIS3L2, missense, het	I	NA	II	DIS3L2, missense, het ABCC2, missense, het KLF10, missense, het ZFHX3, missense, het	
I: primary tumor; Ln: metastatic lymphnode at diagnosis; II; first relapse; III: second relapse; IV, third relapse; letters a, b, c indicate spatially different relapses; s: scar; Het: heterozygous; H/H: Homo/Hemizygous; splice polypyr: splice polypyrimidine tract; splice acc: splice acceptor; splice don: splice donor; NA: Not available. Genes names reported as by HUGO symbol.

Genetic events possibly involved in progression from primary tumor through relapsed disease

We considered a genetic event as possibly involved in tumor progression/relapse in such situations: (1) when the variant was present in the recurrence but absent in the primary tumor; (2) when the variant was present only in a cellular fraction of the primary tumor and uniformly present in the recurrence (= in all the cells analyzed); (3) when the variant was heterozygous in the primary tumor and homo/hemizygous in the recurrence. The BRCA2 variant was an event possibly involved in one relapse in patient no. 3, since it was acquired in one recurrence (this variant was excluded in all the other samples both of the primary tumor and of the other recurrence). MYCN mutation, heterozygous in the only primary tumor sample in which the analysis succeeded, and almost homo/hemizygous in all the fragments of recurrent disease, could be involved in recurrence in patient no. 7. CTNNB1 and LTBP3 variants were likely late events within primary tumor of patient no. 15, considering their low VAF and that CTNNB1 mutation was present in 1/3 primary tumor blocks (data on LTBP3 on primary tumor blocks not investigated by NGS were not available). These variants were consistently present both in first and second recurrences. VCAN and STIL variants were possibly involved in tumor progression, since they were acquired by the second and third recurrence, respectively (and excluded in the two tumor blocks of the primary disease in which sequencing succeeded). Whereas the VAF of CTNNB1 and LTBP3 in the primary tumor were almost the same (and these variants occurred in the same tumor block), considering that the third recurrence displayed the CTNNB1 variant but not the LTBP3 variant, we can speculate that this recurrence originated from a primary disease area bearing the CTNNB1 variant only, thus placing the LTBP3 variant occurring later in the primary disease than the CTNNB1 variant. TP53 variant was involved in the progression of the disease in patient no. 18, since it was present in heterozygosity in one primary tumor sample, but in almost homo/hemizygosity in the remaining two primary tumor samples, in the metastatic lymph node at diagnosis, and in the recurrent sample. MYCN variant was a late occurring event in patient no. 35, since it was present in 1/2 blocks of the primary tumor investigated and in two recurrences, whereas OBSL1 variant was acquired in the same two relapses. CREBBP variant was acquired in the recurrence in patient no. 36, since it was excluded in all the 3 blocks of primary disease investigated. TP53 variant was involved in the progression to a relapsed disease in patient no.111α, being present with a very low VAF in the primary tumor but almost in homo/hemizygosity in the relapse; CHL1 and PRMT7 variants were also acquired in the recurrent disease. Events possibly involved in tumor progression in patient no. 190 were the CHEK2 variant becoming homo/hemizygous in the recurrence, and the ATM variant, acquired in the recurrent disease.

Germline variants

Among the 28 relapsing patients investigated in the present study, 13 (46.4%) carried one or more germline variant. Germline variants already reported in WT patients included those in the TP53 gene (identified in patient no. 3), PALB2 gene (identified in patients no. 7 and no. 12) BLM gene (identified in patients no. 14, no. 34, no. 39α) CHEK2 gene (identified in patients no. 14 and no. 190), BRCA2 gene (identified in patients no. 19 and no. 33), CDC73 gene (identified in patient no. 35), DICER1 gene (identified in patient no. 190), DIS3L2 gene (identified in patient no. 553), MUTYH gene (identified in patient no. 3) and PMS2 gene (identified in patient no. 16).7 To the best of our knowledge, germline variants never reported previously in WT patients include RNASEH2B (identified in patient no. 12, carrying a missense RNASEH2B variant co-occurring with a PALB2 germline missense variant), SDHB and SMARCAL1 (both identified in patient no. 19 carrying a splice donor +1 variant of SDHB and a missense SMARCAL1 variant, co-occurring also with a germline BRCA2 nonsense variant), BBS2 (in patient no. 31 carrying a frameshift BBS2 variant), and CTC1 (in patient no. 33 carrying a frameshift of CTC1 and a germline missense BRCA2 variant). We do not have data on segregation in relatives of the identified carriers, but for patient 19, who inherited the BRCA2 variant from the father and whose family already had genetic counseling.

Targeted sequencing of DGCR8, DROSHA, and MYCN

Targeted sequencing of DGCR8 hotspot mutation was successful in 8/10 cases, and no mutations were found in any of the samples. Targeted sequencing of DROSHA was successful in 9/10 cases, and no mutations were found. Targeted sequencing of MYCN hotspot mutation was successful in 8/9 cases and mutation was found only in 1/2 primary tumor blocks of patient no. 5; however, the mutation was not identified in any of the 4 relapse samples investigated.

Discussion

Whereas the genetic landscape of primary WT has been described in more details,2,3 the genetics of recurrent disease is an ongoing field of investigation, explored in detail only in recent years,5,6,7,9 due to the increasing interest in gaining insights on the genetic events leading to tumor progression/recurrence. The study of recurrent WT should highlight molecular features that may occur as clonal events in the primary disease, and so not of easy identification when studying the primary tumor mass, even more when investigating a single tumor fragment in a genetically heterogeneous disease.8 In fact, a clonal event occurring in the primary WT that is relevant for tumor progression/recurrence is reasonably supposed to be positively selected and thus present in all the cells of recurrent disease. Through multiple samplings of primary and recurrent disease, our study aimed to add evidence to the genetics of recurrent WT, hypothesizing the temporal acquisition of the different anomalies, identifying those acquired de novo or positively selected through the recurrent disease. One final special aim was also to possibly identify therapeutically actionable targets. We previously used a targeted sequencing approach focused on the SIX1 and SIX2 and the miRNAPG DGCR8, DROSHA, and DICER1 genes.5,6 For the present study, we investigated the tumors of 18 patients in which no SIX and/or miRNAPG mutation had been previously found (assuming that in SIX and miRNAPG mutated tumors the possible driver mutations had been already disclosed), and in addition 10 newly collected cases. Overall, we identified 21 variants affecting 12 genes with a role in DNA damage prevention and repair in the relapsing tumor in 12 patients (42.9%). Several genes encoding proteins involved in a variety of DNA repair mechanisms have been identified in the human genome: these genes are involved in homologous recombination, base excision repair, mismatch excision repair, nucleotide excision repair, non-homologous end-joining, or are catalytic subunit of DNA polymerases, or genes defective in diseases associated with sensitivity to DNA damaging agents, or other evolutionary conserved DNA damage response genes (https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html). In particular, we found variants in ATM, which is a master regulator of double-strand DNA break (DSB) signaling and stress responses,10 in BLM, a RECQ helicase involved in DNA replication and repair,11 in BRCA2, involved in DSB repair and homologous recombination (HR),12,13 in CHEK2, involved in activation of DNA repair,14 in CTC1, involved in genome stability maintainance,15 in FANCA, a DNA repair protein,16 in MUTYH, involved in oxidative DNA damage repair,17 in PALB2, which has a crucial role in HR repair,18,19 in PMS2, a component of the DNA mismatch repair system,20 in RNASEH2B, that degrades the RNA of RNA:DNA hybrids,10,21,22 in SMARCAL1, annealing helicase crucial in DNA damage response at stalled replication forks,23,24 and in UBA1, essential for the formation of radiation-induced foci.25 Intriguingly SMARCAL1, in addition to its role in DNA damage response, has been also found, together with BRG1, to transcriptionally upregulate the expression of the miRNAPG DROSHA, DICER1 and DGCR8 in response to doxorubicin induced DNA damage.26 The study from the Children’s Oncology Group found augmented levels of TERT gene (coding for the catalytic subunit of telomerase) expression due to a TERT promoter variant.7 Noteworthy, CTC1 is involved in telomere length homeostasis inhibiting telomerase,27 and thus it is possible to speculate that a cell with a CTC1 frameshift variant will similarly have augmented telomerase activity.

In addition, seven variants affecting 5 genes involved in chromatin modification and regulation were identified in the relapse in 6 patients (21.4%). We found anomalies affecting BMI1, which maintains the transcriptionally repressive state of many genes,28 CDC73, a component of the PAF1 complex which is involved in histone modifications,29 CREBBP, an histone acetyltransferase,30 KMT5B, an histone methyltransferase also involved in DNA repair,31 and PRMT7, an histone methyltransferase.32,33 Anomalies affecting genes involved in transcriptional regulation, such as KLF1034 and PAX8,35 in genes involved in TGF-beta signaling, such as LTBP3,36 SMAD3,37 and ZFHX3,38 in the resistance to several anti-cancer drugs, such as ABCC2,39 in microtubule dynamics and genome integrity, such as OBSL1,40 and in embryonic development and cellular growth and proliferation such as the immediate-early gene STIL,41 were also identified.

Finally, four TP53 variants were found in the relapses in 4 patients (14.3%) with a diffuse anaplastic WT. In 2 cases, TP53 variant occurred together with one or more variant/s of gene/s involved in DNA damage prevention and repair, and in 2 cases together with a variant of a gene involved in chromatin modification and regulation. For each patient in whom a germline variant was identified, the opportunity to offer a genetic counseling was carefully evaluated.

When considering the genes previously proposed as involved in tumor progression/relapse, mainly SIX1, miRNAPG, and MYCN,5,6,7 and their involvement in the relapse in the total of 37 relapsing WT patients that we investigated (28 cases investigated in this study, and additionally 6 cases from Cohorts A1 and 3 cases from Cohort A2 not included in the present analysis5,6), we disclosed miRNAPG mutations in the relapses of 11 patients (29.7%) (6 cases from cohort A1, 3 from cohort A2, 2 cases from the present study), SIX1 mutation in the relapses of 4 patients (10.8%) (1 case from cohort A1, 3 from cohort A2), MYCN focal amplification in 1/8 SNP array investigated relapses (12.5%),5 and MYCN hotspot mutations in the relapses of 2/36 patients (5.6%) (both from the present study).

The present study, mainly focused on relapses in which the involvement of SIX and miRNAPG was excluded, adds the evidence about the involvement in a consistent fraction of cases of genes involved in DNA damage prevention and repair and in chromatin modification and regulation. This finding might suggest that multiple mechanisms underlie WT progression and recurrence, and open the possibility to use targeted therapies in selected cases. It is worthnoting that miRNAPG, in addition to their canonical function, are also involved in DNA damage response,42,43,44 thus making the proportion of WT relapsed cases with a possible impairment of this pathway even higher.

PARP inhibitors are effective when used in tumors with deficiency for homologous recombination.45 In literature, the KT-10 WT patient derived xenografts (with a homo/hemizygous PALB2 frameshift variant) were demonstrated to be highly sensitive to PARP inhibitors,46,47 and in the first phase 1 clinical study of olaparib in pediatric patients with refractory solid tumors, the only WT enrolled patient, who had a WT hemizygous for an ATM variant (displaying 11q loss) showed partial response.48 Biallelic inactivation of genes involved in DNA damage repair were rare in our cases; however, in many tumors we found the co-occurrence of mutations in two different genes involved in DNA damage prevention and repair, suggesting that these WT may have some impairment of this pathway. The pediatric MATCH treatment trial NCT03233204 (https://classic.clinicaltrials.gov/ct2/show/NCT03233204), in which olaparib was used to treat patients with relapsed or refractory advanced solid tumors, including WT, with defects in DNA damage repair genes, required among the inclusion criteria the presence of an actionable mutation, and the majority of patients had somatic or germline monoallelic mutations without somatic loss of heterozygosity or compound heterozygosity.49 Monoallelic mutations in genes involved in DNA damage prevention and repair is what we disclosed in many of our relapsed patients, for whom these targeted therapies might be of interest.

We think that in addition to have a large number of candidate genes involved in its initial development, also the number of genes involved in WT progression and relapse is large.5,6,7,9 Therefore, probably, the best approach to a relapsed child is a personalized intervention after determining the genetic makeup of the relapsed disease/s, being aware that temporally and/or spatially different recurrent diseases may have different genetic make up and that also within the same recurrence, selective pressure continues, generating intratumor heterogeneity. The disclosure of molecular pathways underlying tumor progression could facilitate in specific cases the use of molecular targeted therapies. Incidentally, our data, disclosing numerous germline mutations in the settings of relapsed WT patients, raise, in agreement with others studies, the issue of performing a genetic testing in the settings of a genetic counseling to all children presenting with a WT.50,51,52,53

Limitations of the study

We are aware of the different limits of the present study, which are the number of investigated genes present in the SOPHiA panel (approximately 5000), the cutoff of 15% for the VAF for variants occurring in a single sample of a patient (percentage below which in our hands Sanger validation was not easily possible), and the application of the selection for only biologically relevant genes for tumor development/progression/relapse and for nephrogenesis. These limits are due to the use for the majority of the cases of a finite amount of formalin-fixed paraffin-embedded (FFPE) archival material that imposed methodological restrictions both in the high throughput analysis and in the validation process. However, having validated all the variants reported in the present paper, we are confident about the robustness of our results.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Biological samples	
	
Samples from normal blood or tissue, primary tumor, relapse tumor from patients with Wilms Tumor	Multiple tissue source sites	See This paper (STAR Methods: Experimental model and study participant details)	
	
Critical commercial assays	
	
GeneReadTM DNA FFPE Kit	Qiagen	Cat. No. / ID:56404	
QIAmp DNA Blood Mini Kit	Qiagen	Cat. No. / ID:51104	
DNA Gentra Puregene tissue kit	Qiagen	Cat. No. / ID:158066	
BS-SOPHiA Clinical Exome Solution v2.0 – ILL – R – GL – L01 – 32rx (this code includes: library preparation reagents, protocols, and bioinformatic data analysis by FASTQC and SOPHiA DDM™ platform)	SOPHiA GENETICS	BS0113ILLRGLL01-032	
Kapa HyperPlus Library Preparation Kit	Roche	Kit Code: KK8514	
NextSeq 500/550 v2.5	Illumina	300-cycle high output kit (https://www.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents/nextseq-series-kits-v2-5.html)	
	
Deposited data	
	
Since data on the germline DNA of pediatric patients are also present, the data that support the findings of this study are available from the lead contact upon reasonable request and Data Transfer Agreement.	Lead contact: Daniela Perotti, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milano, Italy	Lead contact: Daniela Perotti daniela.perotti@istitutotumori.mi.it	
	
Oligonucleotides	
	
Custom Primer for variant validation	This paper (Table S3)	N/A	
Primer for DGCR8, DROSHA, MYCN	Spreafico et al.5, Ciceri et al.6,54	N/A	
	
Software and algorithms	
	
SOPHiA DDM™ platform	SOPHiA GENETICS	N/A	
SIFT	Agency for Science, Technology and Research	https://sift.bii.a-star.edu.sg/	
Polyphen-2	Harvard University	http://genetics.bwh.harvard.edu/pph2/index.shtml	
	
Other	
	
Qubit fluorometer	TermoFisher	N/A	
4200 TapeStation	Agilent	N/A	
NextSeq 500 system	Illumina	N/A	

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact Daniela Perotti daniela.perotti@istitutotumori.mi.it.

Materials availability

This study did not generate new unique reagents.

Data and code availability

• Since data on the germline DNA of pediatric patients are also present, the data that support the findings of this study will be shared by the lead contact upon reasonable request and Data Transfer Agreement.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Samples of 28 White children<18 years (boys 13, girls 15) with a relapsing WT diagnosed in Italy were analyzed. In 2016, the centers of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) that had registered WT patients who relapsed in the SIOP-2001 and AIEOP 2003 protocols were asked to participate in a study aimed to characterize the genetic of relapsed WT: 19 paired primary/relapsed tumors were investigated (Cohort A1).6 An additional 8 paired samples from primary and relapsed tumors had been already described in a previous study (Cohort A2).5 For the present study, we chose to focus on tumors in which neither SIX nor miRNAPG mutation had been found, assuming that in those cases possible driver mutations had been already disclosed. This led to a total of 28 relapsing WT cases to investigate: 13 paired primary/relapsed tumors from Cohort A1, 5 paired primary/relapsed tumors from Cohort A2, and additional 10 patients, Cohort A3 (6 paired primary/relapsed and 4 relapses). Clinico-pathological characteristics of investigated patients are depicted in Table S2. Germline material (Peripheral blood leukocytes (PBL)), or FFPE normal kidney (NK) was available for all the patients. The study was approved by the Ethics Committees of Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan (AIEOP protocol) and of Bambino Gesù Children’s Hospital IRCCS, Rome (SIOP protocol) (ethical approval number 47/21), and written informed consent to the use of biological samples and clinical pathological data for research purposes was obtained from the parents or the guardians of all patients.

Method details

Tumor tissue selection

All selected FFPE blocks were centrally revised by expert pediatric pathologists (PC and FDC), and classification was applied along the AIEOP 2003 and SIOP protocols. When possible, multiple FFPE blocks from different tumor areas were selected, in order to better characterize all the different present histologies (i.e., epithelium, blastema, and stroma). If present, anaplastic component was also included. Five 5 μm sections were cut from each selected block and macrodissection was applied when needed, to obtain ≥ 90% of viable tumor cells for each block.

Molecular analyses

FFPE tumor and NK DNA were extracted using the GeneRead™ DNA FFPE Kit (Qiagen, Milan, Italy). PBL DNA was extracted with QIAmp DNA Blood Mini Kit (Qiagen, Milan, Italy). Frozen tissue DNA was extracted using the Gentra Puregene tissue kit (Qiagen, Milan, Italy). Obtained DNA has been quantified with Qubit fluorometer (TermoFisher, Waltham, MA, USA) and DNA integrity has been assessed with 4200 TapeStation, (Agilent Technologies, Santa Clara, CA, USA). For each of the 28 patients, DNA libraries were prepared from germline material (n=28), one primary tumor sample (when available, n=24) one metastatic lymphnode at diagnosis (n=1), and at least one relapse sample (more relapse samples were used for different spatially and/or temporally occurring relapses, n=39). DNA libraries for the SOPHiA Clinical Exome Solution v2.0 (CES_v2) panel (SOPHiA GENETICS, Losanna, Switzerland), which includes approximately 5000 genes, have been prepared starting from genomic DNA with the SOPHiA Solid tumor solution protocol, using the Kapa HyperPlus Library Preparation Kit (Roche, Milan, Italy), for FFPE extracted DNA, and with the SOPHiA Clinical Exome Solution protocol using the SOPHiA DNA Library Prep Kit I for fresh frozen and for PBL extracted DNA. Manufacturer’s guidelines have been followed. DNA libraries have been quantified using a Qubit fluorometer (TermoFisher, Waltham, MA, USA) and assessed for integrity using 4200 TapeStation, (Agilent Technologies, Santa Clara, CA, USA). Libraries were pooled and diluted to 4 nM. Pre-sequencing sample preparation was performed following the manufacture’s instructions (i.e. NextSeq Denature and Dilute Libraries Guide, 15048776 v11). Briefly, 5 μL of diluted library pool was denatured with equal volume of 0.2 N NaOH for 5 minutes at room temperature. After adding 5 μL of 200 mM Tris-HCl pH 7, the denatured library pool was diluited to 20 pM with HT1 buffer. Subsequentially, 117 μL of 20 pM denatured library pool was placed in new 1.5 mL DNA LoBindTube (Eppendorf, Hamburg, Germany) and then 1,183 μL of HT1 was added bringing the library pool to 1.8 pM. The final library was placed on ice until sequencing. Library samples were loaded on the NextSeq 500 system and paired-end sequencing (2 × 150 bp) was carried out using the NextSeq 500/550 v2.5 (Illumina, San Diego, CA, USA) sequencing reagent kit and NextSeq 500 System Guide recommended protocol.

Data processing and bioinformatics analyses

Following sequencing, demultiplexing, and fastq generation, the reads were filtered for quality using FASTQC. The fastq files of each of the 64 tumor (primary and relapse)/normal pairs were uploaded separately to the SOPHiA DDM™ platform (Sophia Genetics, Rolle, Switzerland) to analyze Single Nucleotide Variants (SNVs), and indels for both tumor and normal samples. The reads were aligned against human genome hg19 and raw SNVs/Indels were called by variant caller. A set of filters were applied to the raw SNVs/indels: 1) Variant filtering was set-up according to background noise measured in germline samples. Any variant within these regions with lower variant fraction than the background noise was flagged as high background noise; 2) any variant below 5% variant fraction was flagged as low variant fraction; 3) any variant below 10 alternative read group support was flagged as low molecular_support; 4) any variant with less than 70 molecules for normal sample or less than 100 molecules for tumor sample was flagged as low molecular depth; 5) any variant which falled in 2 bases or 3 bases tandem repeat of minimum 8 bases was flagged as tandem repeat region; 6) any variant which falled in homopolymer region of more than 7 bases was flagged as homopolymer region ; 7) any variant which falled in highly homologous regions such as MUC5B, FLG, DSPP, TYRO3 and FAM8A1 etc was flagged as problematic region; 8) any variant with low quality as determined by statistical test during variant calling was flagged as low quality.

Individual samples text files were manually revised and normal vs. tumor (primary or relapse) comparison performed to identify somatic variants. Due to FFPE starting material, all somatic variants with a VAF ≤5% were excluded. Somatic variants were selected for being: nonsense, frameshift, no start, no stop, splice donor +1 and +2, splice acceptor -1 and -2, missense. Missense variants where further investigated when predicted to be damaging/probably damaging/possibly damaging by at least one predicting program (SIFT, Polyphen), and/or when having in ClinVar database “Pathogenic” or “Likely pathogenic” or “Conflicting interpretations of pathogenicity” or “Uncertain significance” as significance. The function of identified genes and their possible role in the processes of tumor development/progression/relapse and of nephrogenesis were investigated by UniProt and PubMed search, and only genes biologically relevant for these processes were mantained. After consultation with the SOPHiA bioinformatic service, due to the exploratory nature of the use of their pipeline with FFPE material, and thus the necessity to Sanger verify identified variants, single (i.e. in a single sample of a patient) somatic events with a VAF <15% (a threshold below which in our hands it was not possible to visualize the mutant allele) were excluded.

Germline variants were investigated in genes classified as Wilms tumor predisposition genes (BLM, BRCA2, BUB1B, CDC73, DICER1, DIS3L2, GPC3, GPC4, PALB2, PIK3CA, TP53, TRIM37, WT1; the CTR9, REST and TRIM28 genes were not in the panel) and in three additional genes associated with predisposition to adult tumors and with germline mutations identified in patients with WT (CHEK2, MUTYH, PMS2).7,52,55 In addition, germline variants in all the other genes of the panel were further considered when having in ClinVar database “Pathogenic” or “Pathogenic/Likely pathogenic” as clinical significance and when biologically relevant for tumor development/progression/relapse and for nephrogenesis.

For the variants thus selected, custom primers were used and validations were performed by Sanger sequencing (Table S3). Germline variants were verified in PBL or NK only, somatic variants were verified in the sample in which they were identified, then, if validated, in all the tumor samples not investigated by NGS (wherever possible), and also in the samples investigated by NGS in case of low coverage and no detection of the variant in the sample.

The data that support the findings of this study are available from the lead contact author upon reasonable request and Data Transfer Agreement.

Targeted sequencing

The DGCR8 and DROSHA genes were not in the panel used in this study, so targeted sequencing of the DGCR8 hotspot mutation and of the regions of DROSHA mainly involved in mutations in WT was performed in the newly enrolled 10 cases not previously investigated (Cohort A3), as previously described.5,6 MYCN hotspot mutation was sequenced in the 6 cases of Cohort A1 and in the 3 cases of Cohort A2 not included in the panel of cases investigated by NGS, as already described.54

Quantification and statistical analysis

Sanger sequencing analyses of the identified variants were performed in independent duplicate. Statistical analyses were not performed for the present paper.

Supplemental information

Table S1. Genetic data of the investigated WT patients, related to Figure 1 and Table 1

Yellow, green, blue lines indicate germline, primary tumor, and relapse/s investigated by NGS, respectively. Different relapses are indicated with different blues. Sample origin column: germl: germline; I: primary tumor; II; first relapse; III: second relapse; IV, third relapse; letters a, b, c indicate spatially different relapses; LN: metastatic lymphnode at diagnosis. Histological component of FFPE material column: S stromal component; B blastemal component; E epithelial component; ANAPL: anaplasia; DA: diffuse anaplasia. NK: normal kidney.

Table S2. Description of patients characteristics, related to STAR Methods

RT: Radiotherapy; DFS: disease-free survival; I: primary tumor; II: first relapse; III: second relapse; IV: third relapse M: male; F: female; N: no; Y: yes; NA: not available; R: Right; L: Left; WT: Wilms tumor; ∗: SIOP 2001.

Table S3. Custom primers used for Sanger sequencing validations, related to STAR Methods

Acknowledgments

This work was supported by Fondazione Bianca Garavaglia, Busto Arsizio, Italy and Ministry of Health - Ricerca Corrente fundings. Authors thank Mrs. Donata Penso, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan Italy, for technical support, Dr. Mara Colombo, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy, for helpful discussion on BRCA variants, Dr. Yvan Wenger and Dr. Melissa Wong, SOPHiA Genetics for bioinformatic support. Graphical abstract was created with BioRender.

Author contributions

Conceptualization: S.C., P.C., F.S., and D.P.; methodology: S.C., A.B., L.D.C., G.M., F.D.C., P.C., and D.P.; investigation: S.C., A.B., L.D.C., and D.P.; resources: A.S., G.G., L.B., M.C., C.C., S.S., P.Q., C.M.C., P.V., F.D.C., and D.P.; data curation: S.C., L.D.C., and D.P.; writing – original draft: S.C., A.B., L.D.C., P.C., F.S., and D.P.; writing – review and editing: A.S., G.G., L.B., M.C., C.C., S.S., P.Q., C.M.C., P.V., G.M., and F.D.C.; visualization: S.C. and D.P.; supervision: F.S. and D.P.; funding acquisition: D.P.

Declaration of interests

The authors declare no competing interest.

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110684.
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