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Sci Rep
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Scientific Reports
2045-2322
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10.1038/s41598-024-71667-x
Article
The coexistence of a BRCA2 germline and a DICER1 somatic variant in two first-degree cousins suggests their potential synergic effect
Del Baldo Giada 12
Mastronuzzi Angela angela.mastronuzzi@opbg.net

1
Cipri Selene 1
Agolini Emanuele 3
Matraxia Marta 3
Novelli Antonio 3
Cacchione Antonella 1
Serra Annalisa 1
Carai Andrea 4
Boccuto Luigi 5
Colafati Giovanna Stefania 6
Di Paolo Pier Luigi 6
Miele Evelina 1
Barresi Sabina 7
Alaggio Rita rita.alaggio@opbg.net

7
Rossi Sabrina 7
Giovannoni Isabella 7
1 https://ror.org/02sy42d13 grid.414125.7 0000 0001 0727 6809 Pediatric Haematology and Oncology, and Cell and Gene Therapy Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
2 https://ror.org/02be6w209 grid.7841.a Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
3 https://ror.org/02sy42d13 grid.414125.7 0000 0001 0727 6809 Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
4 https://ror.org/02sy42d13 grid.414125.7 0000 0001 0727 6809 Neurosurgery Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
5 https://ror.org/037s24f05 grid.26090.3d 0000 0001 0665 0280 School of Nursing, College of Behavioral, Social and Health Sciences, Clemson University, Clemson, SC USA
6 https://ror.org/02sy42d13 grid.414125.7 0000 0001 0727 6809 Imaging Department, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
7 https://ror.org/02sy42d13 grid.414125.7 0000 0001 0727 6809 Pathology Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
13 9 2024
13 9 2024
2024
14 2143517 5 2024
29 8 2024
© The Author(s) 2024
2024
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Cancer predisposition syndromes are recognized in about 10% of pediatric malignancies with several genes specifically involved in a subset of pediatric tumors such as DICER1, in pleuropulmonary blastoma, cystic nephroma, and brain sarcomas. By contrast, the role of BRCA1/2 in pediatric cancer predisposition is still under investigation. We present two cases of young first-degree cousins, both carrying a germline BRCA2 variant and developing tumors characterized by somatic DICER1 mutations. Patient 1 presented with a cystic nephroma harboring a somatic DICER1 variant (p.Asp1810Tyr), while patient 2 had a primary intracranial DICER1-mutated sarcoma showing a distinct somatic DICER1 variant (p.Asp1709Glu) as well as biallelic inactivation of TP53 (p.Val173Leu, VAF 91%) and APC (p.Ile1307Lys, VAF 95%) and a pathogenic variant in KRAS (p.Gln61His). Both patients carried the same germline BRCA2 variant (p.Arg2842Cys) of unknown significance. The same variant was found in the mother of patient 2 and in the father of patient 1, who are siblings. A homologous recombination deficiency signature was not identified in any of the two tumors, possibly suggesting a reduction of BRCA2 activity. The association of BRCA2 and DICER1 variants in our cases hints at a potential cooperative role in cancer pathogenesis. Further studies are warranted to elucidate the interplay between BRCA1/2 and DICER1 variants and their implications for cancer predisposition and treatment in pediatric patients.

Keywords

Cancer predisposition syndrome
Pediatric oncology
BRCA genes
DICER1
Cystic nephroma
Primary intracranial DICER1-mutated sarcoma
Subject terms

Cancer genetics
Paediatric cancer
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Cancer predisposition syndromes (CPSs) are described in about 10% of overall pediatric cancers1. The role of genetic factors influencing the onset and progression of cancer is gradually being identified and understood with increasing precision and translational efficacy. The best-known cancer-predisposition genes (CPGs) that play a role during pediatric age are ALK, DICER1, ELP1, GATA2, NF1, PAX5, RB1, RET, RUNX1, SDHx, SMARCB1, SUFU, TP53, and WT11.

Among the genes associated with tumor predisposition in children, DICER1 is one of the most explored. DICER1 encodes an enzyme involved in the cytoplasmic microRNA (miRNA) processing. miRNAs play crucial roles in post-transcriptional gene regulation, and alterations in their processing can lead to dysregulation of gene expression, which in turn can contribute to tumorigenesis2,3. Notably, the Dicer protein is also linked to the DNA damage response and genome integrity maintenance, as it is involved in chromatin de-condensation during nucleotide excision repair4, in small noncoding RNA accumulation at the level of DNA double-strand breaks (i.e., DNA damage response RNA)5–8 and in resolution of three-stranded nucleic acid structures (i.e., R-loops) driving transcription-associated genomic instability9. DICER1 syndrome is a pediatric cancer predisposition syndrome typically caused by heterozygous germline DICER1 loss-of-function variants accompanied by somatic second hit variants on the other allele within the sequence encoding the RNase IIIb domain. Individuals affected by DICER1 syndrome are predisposed to benign and malignant neoplasms in multiple organ systems, such as pleuropulmonary blastoma, cystic nephroma, and Sertoli–Leydig cell tumor of the ovary. Additionally, DICER1 mutations have been reported in other pediatric cancers, including Wilms tumor, embryonal rhabdomyosarcoma10, and certain rare types of brain tumors11,12. Nevertheless, sporadic tumors in which two somatic DICER1 variants are identified have been described in the absence of an identified germline alteration2.

By contrast, BRCA1 and BRCA2 are established autosomal dominant predisposition genes for breast, ovarian, pancreatic, and prostate cancers in adults13,14, while their role in childhood cancer is poorly understood.

BRCA1 and BRCA2 are involved in DNA damage response, specifically in the homologous recombination repair pathway. Cells with pathogenic variants in BRCA genes are error-prone at the DNA level and accumulate mutations leading to genome instability, showing a typical homologous recombination deficiency (HRD) signature. Tumors arising in this context show a “BRCAness phenotype”15 and are likely to respond to Poly (ADP-ribose) polymerase inhibitors (PARPis)16.

In the pediatric population, biallelic mutations in BRCA2 can lead to clinical manifestations, whereas alterations in BRCA1 are rare, typically presenting as the recessive disorder Fanconi anemia17–19. Children are not routinely tested for BRCA1 and BRCA2 variants unless Fanconi anemia is suspected. However, the increasing use of high-throughput technologies in clinical laboratories and the agnostic integration of blood and tumor sequencing has recently brought to light the role of germline mutations in BRCA genes and other DNA damage repair genes in several other types of pediatric cancers (osteosarcoma, Ewing sarcoma, neuroblastoma, medulloblastoma, acute lymphoblastic leukemia)20.

Here, we report on two young first-degree cousins carrying a germline BRCA2 variant and developing different solid tumor types, i.e., cystic nephroma and primary intracranial sarcoma, both characterized by somatic inactivation of DICER1. This report enables a better understanding of the biological bases of these pediatric tumors, allowing for more effective management of these oncological conditions and opening new opportunities for the development of targeted therapies and preventive strategies.

Results

A 14-month-old girl (patient 1) was admitted to our hospital with a palpable abdominal mass on the right side without other relevant symptoms. No anomalies were detected during pregnancy. An abdominal ultrasound revealed a large left renal mass, causing a compression of the excretory tract and distension of the pelvic cavities (Fig. 1). Tumor marker tests were performed and showed elevated renin values (185 μIU/mL with normal values of 2.8–39.9); other markers, such as alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), chromogranin A (CgA) and carcinoembryonic antigen (CEA) were negative. Chemotherapy was started according to the SIOP 2001 protocol; then, the patient underwent a left nephrectomy. On histological examination, the tumor had the characteristics of a cystic nephroma (Fig. 2). Sanger sequencing analysis showed the presence of a somatic pathogenic variant c.5428G > T (p.Asp1810Tyr) in the RNase IIIb domain of DICER1 gene (NM_177438.3). Currently, the patient is in follow-up and is in complete remission of the disease at 24 months from diagnosis.Fig. 1 Axial contrast-enhanced CT (a) and axial T2WI MRI (b) show a large cystic mass arising from the left kidney with multiple septations.

Fig. 2 At histological examination, the lesion of patient 1 consisted of multiple cysts lined by a cubic epithelium and separated by fibrous septa containing occasional abortive tubules.

Six months later, the first-degree cousin of patient 1, a 4-year-old girl (patient 2), was admitted to our hospital for a history of headache and vomiting associated with lethargy. Computed tomography (CT) showed a right frontoparietal lesion with signs of intra-tumoral hemorrhage (Fig. 3). For the progressive deterioration of clinical conditions and neurological status, the patient underwent urgent neurosurgical resection of the tumor. Histological examination revealed a malignant spindle cell sarcoma with focal nuclear pleomorphism and rare eosinophilic globules (Fig. 4A,B). Immunophenotypic features showed overexpression of p53 and loss of ATRX, mosaics from loss of H3K27me3 and focal expression of SALL4 and PRAME (Fig. 4C–G). The findings overall suggested a primary intracranial DICER1-mutated sarcoma, which was further confirmed by DNA methylation profiling and the identification of a somatic pathogenic variant c.5127T > A (p.Asp1709Glu) in the RNase IIIb domain of DICER1 gene (NM_177438.3). The patient underwent a second neurosurgery, confirming gross total resection of the tumor. The cerebrospinal fluid (CSF) was negative for neoplastic cells, and postoperative imaging showed no metastatic lesions or extracranial localization. The patient was started on chemotherapy with a 4-cycle regimen of ifosfamide, carboplatin, etoposide (ICE)21. Subsequently, she underwent two cycles of high-dose chemotherapy with Thiotepa followed by autologous stem cell reinfusion and focal proton beam therapy (PBT). She is currently in complete remission at 18 months from the diagnosis.Fig. 3 Noncontrast (a) and contrast-enhanced (b) axial CT images show a large right fronto-parietal intracerebral hemorrhagic (ICH) lesion with cerebral herniation. No areas of contrast-enhancement or signs of vascular malformation are detected. The regions of hypo- and iso-attenuation (arrows) within the hyperattenuated ICH are suggestive of hyperacute/active hemorrhage, and the consensual presence of substantial surrounding edema raises suspicion of an underlying tumor.

Fig. 4 At histological examination, the lesion of patient 2 consisted of fascicles of spindle cells with hyperchromatic nuclei, brisk mitoses (A), focal nuclear pleomorphism and rare eosinophilic globules (B). The tumor showed overexpression of p53 (C), loss of ATRX (D), overexpression of SALL4 (E) and PRAME (F) and mosaiciform loss of H3K27me3 (G).

Germline next-generation sequencing (NGS) analysis excluded the presence of variants in the DICER1 gene and showed that both patients carried a heterozygous missense variant c.8524C > T (p.Arg2842Cys) in the BRCA2 gene. The same variant was found in the mother of patient 2 and in the father of patient 1, who are siblings (Fig. 5). The variant is classified as a variant of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) and functional assay demonstrates that this variant only partially complemented HR efficiency compared with wild-type BRCA222. In patient 1, a heterozygous splicing variant c.250 + 1G > T (p.?) in the FANCC gene (NM_ 000136.3) with maternal segregation was also identified. This variant is considered as likely pathogenic and has never been reported in literature. In patient 2, an additional heterozygous missense variant c.3920T > A (p.Ile1307Lys) in the APC gene (NM_000038.6) with maternal segregation was recognized. This alteration is considered a risk allele for colorectal cancer and is not known to be associated with polyposis23. Family history reported only one great-grandfather of both patients (not analyzed) with gastric cancer and a maternal grandmother of patient 2 with lung cancer (Fig. 5).Fig. 5 Pedigree of the family. An asterisk indicates the individuals who were tested by DNA sequencing. Detection of the variants c.8524C > T in the BRCA2 gene and c.3920T > A in the APC gene by genetic tests is indicated by the plus sign.

Given the insights from the germline data, somatic NGS analysis was performed through the OncoDeep kit on DNA extracted from formalin-fixed paraffin-embedded tumor tissues. Both samples showed low tumor mutational burden and stable MSI status. The analysis confirmed the PCR results on DICER1 variants, unveiling a variant allele frequency (VAF) of 24% and 46% in patient 1 and 2 tumors, respectively. The germline heterozygous BRCA2 variant c.8524C > T (p.Arg2842Cys) was also evident in both tumors. However, an HRD signature was not identified in any of the samples. Of note, the primary intracranial sarcoma (patient 2 tumor) harbored additional missense pathogenic/likely pathogenic variants: c.517G > C (p.Val173Leu) in TP53 (NM_000546.6) with a VAF of 91%, c.3920T > A (p.Ile1307Lys) in APC (NM_000038.6) with a VAF of 95% and c.183A > C (p.Gln61His) in KRAS (NM_033360.4) with a VAF of 69% as well as multiple loss of heterozygosity involving other DNA repair genes (i.e. APC, TP53, CHEK2, RAD21, FANCF). Germline and somatic data are summarized in Table 1.Table 1 Molecular results from germline and somatic analysis.

	Age at diagnosis	Tumor biomarkers	Gene	Cat	cDNA variant	Amino acid variant	Biological impact	Germline variants	Somatic variants	
TMB score	MSI score	HRD score	VAF	Inheritance	VAF	CNA	
Patient 1	14 months-old	Low (1.7)	Stable (4%)	Negative (23)	BRCA2	SNV	NM_000059.4:c.8524C > T	p.Arg2842Cys	LP	50%	Paternal	51%	–	
DICER1	SNV	NM_177438.3:c.5428G > T	p.Asp1810Tyr	LP	–		24%	–	
FANCC	SNV	NM_000136.3:c.250 + 1G > T	–	LP	50%	Maternal	47%	–	
BRCA2	SNV	NM_000059.4:c.8524C > T	p.Arg2842Cys	LP	50%	Maternal	46%	–	
DICER1	SNV	NM_177438.3:c.5127T > A	p.Asp1709Glu	LP	–		46%	–	
KRAS	SNV	NM_033360.4:c.183A > C	p.Gln61His	P	–		69%	–	
TP53	LOH	-	–	LP	–		–	1	
Patient 2	4 years-old	Low (1.7)	Stable (3.7%)	Negative (18)	TP53	SNV	NM_000546.6:c.517G > C	p.Val173Leu	LP	–		91%	–	
APC	SNV	NM_000038.6:c.3920T > A	p.Ile1307Lys	LP	50%	Maternal	95%	–	
CHEK2	LOH	-	–	LP	–		–	1	
RAD21	LOH	-	–	LP	–		–	1	
FANCF	LOH	-	–	LP	–		–	1	
TMB Tumor mutational burden, MSI Microsatellite instability, HRD Homologous recombination deficiency. SNV Single nucleotide variant, LOH Loss of heterozygosity, LP Likely pathogenic variant, VUS Variant of uncertain significance, P Pathogenic variant, VAF Variant allele frequency, CAN Copy number alteration.

Based on the genetic testing results, both patients will undergo surveillance for breast and ovarian cancer as for clinical practice guidelines published24. Moreover, for patient 2 with a likely pathogenic variant in APC, the recommendations for APC conditions reported in the literature will be followed25. Such recommendations will also be applied to family members in whom these variants have been found.

Discussion

In the era of precision medicine, genetic profiling has become essential in pediatric oncology26. The identification of genetic alterations in childhood cancers allows both better prognostic definition and the development of targeted and personalized therapeutic strategies27. Mutations of BRCA1 and BRCA2 were identified in 1993 and 1994 and are associated with an increased risk of developing a wide spectrum of tumors during adulthood, typically breast and ovarian cancer, but also several other histotypes, such as pancreatic and prostate cancer and melanoma28. A role of BRCA2 in childhood cancer predisposition has more recently emerged, and, remarkably, BRCA2 has proved to be the second most mutated gene after TP53 in the Pediatric Cancer Genome Project (PCGP)29,30. The spectrum of cancers observed in children carrying germline BRCA2 mutations described to date includes leukemia, medulloblastoma, neuroblastoma, osteosarcoma, and Ewing sarcoma20.

Here, we report on two young first-degree cousins, both developing a solid tumor with a distinct somatic mutation of DICER1. Patient 1 was affected by a cystic nephroma, a tumor typically considered pathognomonic of DICER1 syndrome31. Patient 2 presented with a primary intracranial DICER1-mutated sarcoma, which may be sporadic or occur in the context of DICER1 syndrome, with an isolated case described in an individual affected by neurofibromatosis type 132. We were able to document a DICER1 pathogenic mutation affecting the RNase IIIb domain in each tumor, namely p.Asp1810Tyr (patient 1 tumor) and p.Asp1709Glu (patient 2 tumor); the VAF of these variants (24% and 46% respectively) did not indicate a biallelic inactivation of DICER1 in the tumors suggesting that Dicer1 activity was not completely abrogated. Intriguingly, the patients did not carry a DICER1 germline mutation but shared the p.Arg2842Cys VUS in BRCA2. This variant is reported in a homozygous condition in primary ovarian insufficiency and was demonstrated to exhibit a reduced DSB-induced HR efficiency22. To shed light on the implication of the BRCA2 p.Arg2842Cys variant in our cases, we sought to investigate the presence of the HRD signature in both tumors. However, we failed to demonstrate a BRCAness signature. It has to be highlighted that currently, no clinically validated diagnostic tests exist for determining BRCAness in pediatric cancer. The threshold we used in the analysis was specifically designated for this signature in adult ovarian and breast cancer and may not be accurate for the evaluation of other tumor entities occurring in children33. Hence, the possibility of a reduction of BRCA2 activity could not be completely excluded. On the other hand, both patients carried germline likely pathogenic variants on genes that are involved in cancer predisposition syndromes. Patient 1 harbored the splice variant c.250 + 1G > T (p.?) on the FANCC gene. The FANCC protein is present in the Fanconi anemia pathway and is involved in the DNA repair pathway, especially for DNA interstrand crosslink (ICL) repair34,35, both in metabolic disorders and protection against oxidative stress-induced apoptosis36,37. Germline FANCC mutations are linked to familial breast cancer and rarely have been associated with renal tumor, and are not currently linked to any hereditary renal cancer predisposition syndromes38. Patient 2 also carried a likely pathogenic heterozygous APC missense variant, i.e., c.3920T > A (p.Ile1307Lys), associated with an increased risk of developing colon cancer. Germline pathogenic variants of APC cause familial adenomatous polyposis (FAP), an inherited colorectal cancer syndrome characterized by the development of hundreds to thousands of adenomas throughout the large bowel. Patients with FAP are also predisposed to developing other neoplasms, including desmoid tumors or aggressive fibromatosis, which are rare mesenchymal tumors of intermediate malignant potential39. Notably, the somatic VAF of the APC mutation was 96%, pointing to biallelic inactivation of the APC gene in the primary intracranial DICER1-mutated sarcoma. These data overall suggest a possible contribution of APC mutation to the pathobiology of this tumor.

To our knowledge, the coexistence of BRCA2 and DICER1 variants has not been reported up to now. The findings in the two patients of two different variants in the somatic hotspot region of DICER1 may not be a simple coincidence, considering also the histotypes, which are typical of DICER1 syndrome and the absence of DICER1 biallelic inactivation in the tumors. Some DNA repair factors have been described to have RNA-binding capacities, and screens investigating DNA damage repair mechanisms have identified RNA-binding proteins as a major group of novel factors involved in DNA repair40. Moreover, it is described that RNA is an important component of the DNA damage response and that it is required to maintain the fidelity of repair. Theoretically, mutations in components of RNA-dependent DNA repair could result in an increased mutation rate, thereby promoting carcinogenesis and cancer progression40. It is of note that the RNA endonuclease Dicer, besides its major role in the miRNA biogenesis, is a crucial player in the DNA damage response and genome integrity maintenance and is required to propagate Double-strand DNA repair4–9 Depletion of RNA endonuclease results in deficient recruitment of repair factors to the damaged site, and reported assays show a significant reduction in both HR and non-homologous end-joining repair efficiency, comparable with that seen after BRCA1 and 53BP1 depletion8,41. Based on our findings, it is tempting to speculate that BRCA2 and DICER1 variants may have a synergic effect resulting in the simultaneous impairment of relevant DNA repair pathways.

Conclusion

We reported for the first time the occurrence of two neoplasms typical of DICER-tumor predisposition syndrome in two patients carrying the same germline BRCA2 variant and a somatic DICER1 mutation. These findings suggest a potential cooperative tumorigenic role of DICER1 and BRCA2, both implicated in DNA repair pathways. Furthermore, this study demonstrates the importance of studying BRCA variants in the pediatric cancer patient population for the potential screening, risk-reductive interventions, and family planning options currently available for relatives of children carrying BRCA mutations. Extending the analysis of these genes to the pediatric population could provide relevant insights correlated to a possible predisposition to cancer.

Methods

Patients

Written informed consents for genetic analysis were obtained from/for all the participants. The study was approved by Institutional Review Board (IRB) of Bambino Gesù Children’s Hospital (RAP-2024-002). All experimental work was carried out according to the declaration of Helsinki.

Germline NGS analysis

Genomic DNA was extracted using the DNA Blood Mini Kit (Qiagen, Hilden, NW, Germany) according to the manufacturer's instructions from circulating leukocytes of peripheral blood samples.

DNA quantification was performed using a Qubit fluorimeter (Life Technologies, Carlsbad, California, USA) with the dsDNA HS Assay kit following the manufacturer's instructions. The genetic analysis was performed through Next Generation Sequencing (NGS) by using a custom clinical exome panel (Twist Bioscience, South San Francisco, CA, USA) that contained more than 8500 genes, including the ones involved in cancer-predisposition syndromes, on NovaSeq 6000 platforms (Illumina, San Diego, CA, USA). The genes were analyzed according to clinical indication for patient 1: BAP1, BRCA2, CDC73, CHEK2, DICER1, DIS3L2, EPCAM, FBXW7, FH, FLCN, MET, MITF, MLH1, MSH2, MSH6, PIK3CA, PMS2 (only exons 1–10), PTEN, SDHA, SDHB, SDHC, SDHD, SMARCB1, TP53, TRIM28, TSC1, TSC2, VHL, WT1 and for patient 2: APC, BLM, CDKN1C, DICER1, EPCAM, EXT1, EXT2, FH, HRAS, KIT, MLH1, MSH2, MSH6, NBN, NF1, PDGFRA, PMS2 (only exons 1–10), PRKAR1A, PTCH1, RB1, RECQL4, SDHA, SDHB, SDHC, SDHD, SUFU, TP53, WRN, CDKN2A, POT1, PTCH2, TINF2, TSC1, TSC2, BRCA2. The BaseSpace pipeline (Illumina, https://basespace.illumina.com/) and the GeneYX software (LifeMap Sciences) were used for the variant calling and annotating variants, respectively. Sequencing data were aligned to the hg19 human reference genome. Variants were examined for coverage and Qscore (minimum threshold of 30) and visualized by the Integrative Genome Viewer (IGV).

In addition, the reclassification of the variants of uncertain significance was based on the use of the database of variants of the general population—gnomAD (https://gnomad.broadinstitute.org/); the database of correlation between variants and phenotypes—ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/); the database of variants described in the literature—HGMD Professional (http://www.hgmd.org), the scientific literature reported on PubMed; the search engine of human genomic variants—Varsome (https://varsome.com/) and the pathogenicity prediction algorithms.

Somatic NGS analysis

DNA from cystic nephroma and intracranial sarcoma were extracted from formalin-fixed paraffin-embedded tumor tissue using Maxwell CSC instrument (Promega, Madison, USA) with the Maxwell RSC DNA FFPE kit (Promega, Madison, USA) according to the manufacturer’s protocol; DNA concentrations were measured on a Qubit 2.0 Fluorometer (Thermofisher Scientific, Waltham, USA) using the Qubit dsDNA High Sensitivity.

Next-generation sequencing was performed through the OncoDeep kit (OncoDNA). The OncoDeep assay is a comprehensive genomic profiling performed in NGS and targeting 638 cancer-relevant genes. The assay detects indels, small nucleotide variants (SNVs), splice variants and copy-number/structural variations in several genes and also provides complex genomic signatures: tumor mutation burden (TMB), microsatellite status (MSI), and homologous recombination deficiency (HRD) status.

The OncoKDM v24.0.2 software (OncoDNA, Gosselies, Belgium) was used for the variant calling and annotating variants. Sequencing data were aligned to the hg19 human reference genome. Variants were examined for coverage and Qscore (minimum threshold of 150) and visualized by the Integrative Genome Viewer (IGV).

Variant classification was performed following the AMP/ASCO guidelines42.

Acknowledgements

The authors thank Megan Eckley for helping in the English final version.

Author contributions

G.D.B., S.C., I.G., and S.R. wrote the manuscript. I.G., S.C., and E.A. cured the literature research focusing on the genetics aspect. S.R. provided the histological and immunohistochemistry data. E.A., M.M., I.G., S.B. and E.M. provided the genetic data. A.C. (Antonella Cacchione) and A.R. contributed to clinical data curation. G.S.C. and P.L.D.P. contributed to radiological data. A.M., L.B., A.N., A.C. (Andrea Carai), and R.A. critically revised the manuscript for intellectual content. All authors finally approved the version to be published and agreed to be accountable for all aspects of the work to ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available in clinVAR repository (submission ID somatic variant: SUB14648587, submission ID germline variant: SUB14646194, SCV005093812; SUB14646177, SCV005093810; SUB14646132, SCV005093809).

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All participants gave written informed consent, according to CARE guidelines and in compliance with the Declaration of Helsinki principles.

Consent for publication

Code of approval: RAP-2024-002.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Giada Del Baldo and Angela Mastronuzzi.

These authors jointly supervised this work: Sabrina Rossi and Isabella Giovannoni.
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References

1. Kratz CP Predisposition to cancer in children and adolescents Lancet Child Adolesc. Health. 2021 5 2 142 154 10.1016/S2352-4642(20)30275-3 33484663
Kratz, C. P. et al. Predisposition to cancer in children and adolescents. Lancet Child Adolesc. Health. 5(2), 142–154 (2021).33484663 10.1016/S2352-4642(20)30275-3
2. Foulkes WD Priest JR Duchaine TF DICER1: Mutations, microRNAs and mechanisms Nat. Rev. Cancer. 2014 14 10 662 672 10.1038/nrc3802 25176334
Foulkes, W. D., Priest, J. R. & Duchaine, T. F. DICER1: Mutations, microRNAs and mechanisms. Nat. Rev. Cancer. 14(10), 662–672 (2014).25176334 10.1038/nrc3802
3. Kumar MS Lu J Mercer KL Golub TR Jacks T Impaired microRNA processing enhances cellular transformation and tumorigenesis Nat. Genet. 2007 39 5 673 677 10.1038/ng2003 17401365
Kumar, M. S., Lu, J., Mercer, K. L., Golub, T. R. & Jacks, T. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat. Genet. 39(5), 673–677 (2007).17401365 10.1038/ng2003
4. Chitale S Richly H DICER and ZRF1 contribute to chromatin decondensation during nucleotide excision repair Nucleic Acids Res. 2017 45 10 5901 5912 10.1093/nar/gkx261 28402505
Chitale, S. & Richly, H. DICER and ZRF1 contribute to chromatin decondensation during nucleotide excision repair. Nucleic Acids Res. 45(10), 5901–5912 (2017).28402505 10.1093/nar/gkx261
5. Lee HC qiRNA is a new type of small interfering RNA induced by DNA damage Nature. 2009 459 7244 274 277 10.1038/nature08041 19444217
Lee, H. C. et al. qiRNA is a new type of small interfering RNA induced by DNA damage. Nature. 459(7244), 274–277 (2009).19444217 10.1038/nature08041
6. Francia S Site-specific DICER and DROSHA RNA products control the DNA-damage response Nature. 2012 488 7410 231 235 10.1038/nature11179 22722852
Francia, S. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature. 488(7410), 231–235 (2012).22722852 10.1038/nature11179
7. Michalik KM Böttcher R Förstemann K A small RNA response at DNA ends in Drosophila Nucleic Acids Res. 2012 40 19 9596 9603 10.1093/nar/gks711 22848104
Michalik, K. M., Böttcher, R. & Förstemann, K. A small RNA response at DNA ends in Drosophila. Nucleic Acids Res. 40(19), 9596–9603 (2012).22848104 10.1093/nar/gks711
8. Wei W A role for small RNAs in DNA double-strand break repair Cell. 2012 149 1 101 112 10.1016/j.cell.2012.03.002 22445173
Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell. 149(1), 101–112 (2012).22445173 10.1016/j.cell.2012.03.002
9. Camino LP DICER ribonuclease removes harmful R-loops Mol. Cell. 2023 83 20 3707 3719.e5 10.1016/j.molcel.2023.09.021 37827159
Camino, L. P. et al. DICER ribonuclease removes harmful R-loops. Mol. Cell. 83(20), 3707-3719.e5 (2023).37827159 10.1016/j.molcel.2023.09.021
10. Robertson JC Jorcyk CL Oxford JT DICER1 syndrome: DICER1 mutations in rare cancers Cancers (Basel). 2018 10 5 143 10.3390/cancers10050143 29762508
Robertson, J. C., Jorcyk, C. L. & Oxford, J. T. DICER1 syndrome: DICER1 mutations in rare cancers. Cancers (Basel). 10(5), 143 (2018).29762508 10.3390/cancers10050143
11. de Kock L Priest JR Foulkes WD Alexandrescu S An update on the central nervous system manifestations of DICER1 syndrome Acta Neuropathol. 2020 139 4 689 701 10.1007/s00401-019-01997-y 30953130
de Kock, L., Priest, J. R., Foulkes, W. D. & Alexandrescu, S. An update on the central nervous system manifestations of DICER1 syndrome. Acta Neuropathol. 139(4), 689–701 (2020).30953130 10.1007/s00401-019-01997-y
12. Kamihara J DICER1-associated central nervous system sarcoma in children: Comprehensive clinicopathologic and genetic analysis of a newly described rare tumor Mod. Pathol. 2020 33 10 1910 1921 10.1038/s41379-020-0516-1 32291395
Kamihara, J. et al. DICER1-associated central nervous system sarcoma in children: Comprehensive clinicopathologic and genetic analysis of a newly described rare tumor. Mod. Pathol. 33(10), 1910–1921 (2020).32291395 10.1038/s41379-020-0516-1
13. Neuhausen S Recurrent BRCA2 6174delT mutations in Ashkenazi Jewish women affected by breast cancer Nat. Genet. 1996 13 1 126 128 10.1038/ng0596-126 8673092
Neuhausen, S. et al. Recurrent BRCA2 6174delT mutations in Ashkenazi Jewish women affected by breast cancer. Nat. Genet. 13(1), 126–128 (1996).8673092 10.1038/ng0596-126
14. Pritchard CC Inherited DNA-repair gene mutations in men with metastatic prostate cancer N. Engl. J. Med. 2016 375 5 443 453 10.1056/NEJMoa1603144 27433846
Pritchard, C. C. et al. Inherited DNA-repair gene mutations in men with metastatic prostate cancer. N. Engl. J. Med. 375(5), 443–453 (2016).27433846 10.1056/NEJMoa1603144
15. Turner N Tutt A Ashworth A Hallmarks of «BRCAness» in sporadic cancers Nat. Rev. Cancer. 2004 4 10 814 819 10.1038/nrc1457 15510162
Turner, N., Tutt, A. & Ashworth, A. Hallmarks of «BRCAness» in sporadic cancers. Nat. Rev. Cancer. 4(10), 814–819 (2004).15510162 10.1038/nrc1457
16. Mark LR Terp SK Krarup HB Thomassen M Pedersen IS Bøgsted M Homologous recombination deficiency detection algorithms: A systematic review Cancers (Basel). 2023 15 23 5633 10.3390/cancers15235633 38067337
Mark, L. R. et al. Homologous recombination deficiency detection algorithms: A systematic review. Cancers (Basel). 15(23), 5633 (2023).38067337 10.3390/cancers15235633
17. D’Andrea AD Grompe M Molecular biology of Fanconi anemia: Implications for diagnosis and therapy Blood. 1997 90 5 1725 1736 10.1182/blood.V90.5.1725 9292505
D’Andrea, A. D. & Grompe, M. Molecular biology of Fanconi anemia: Implications for diagnosis and therapy. Blood. 90(5), 1725–1736 (1997).9292505 10.1182/blood.V90.5.1725
18. Howlett NG Biallelic inactivation of BRCA2 in Fanconi anemia Science. 2002 297 5581 606 609 10.1126/science.1073834 12065746
Howlett, N. G. et al. Biallelic inactivation of BRCA2 in Fanconi anemia. Science. 297(5581), 606–609 (2002).12065746 10.1126/science.1073834
19. Sawyer SL Biallelic mutations in BRCA1 cause a new Fanconi anemia subtype Cancer Discov. 2015 5 2 135 142 10.1158/2159-8290.CD-14-1156 25472942
Sawyer, S. L. et al. Biallelic mutations in BRCA1 cause a new Fanconi anemia subtype. Cancer Discov. 5(2), 135–142 (2015).25472942 10.1158/2159-8290.CD-14-1156
20. Walsh MF Germline BRCA2 mutations detected in pediatric sequencing studies impact parents’ evaluation and care Cold Spring Harb. Mol. Case Stud. 2017 3 6 a001925 10.1101/mcs.a001925 28655807
Walsh, M. F. et al. Germline BRCA2 mutations detected in pediatric sequencing studies impact parents’ evaluation and care. Cold Spring Harb. Mol. Case Stud. 3(6), a001925 (2017).28655807 10.1101/mcs.a001925
21. Lafay-Cousin L Successful treatment of primary intracranial sarcoma with the ICE chemotherapy regimen and focal radiation in children J. Neurosurg. Pediatr. 2016 17 3 298 302 10.3171/2015.6.PEDS14709 26588458
Lafay-Cousin, L. et al. Successful treatment of primary intracranial sarcoma with the ICE chemotherapy regimen and focal radiation in children. J. Neurosurg. Pediatr. 17(3), 298–302 (2016).26588458 10.3171/2015.6.PEDS14709
22. Caburet, S. et al. Homozygous hypomorphic BRCA2 variant in primary ovarian insufficiency without cancer or Fanconi anaemia trait. J. Med. Genet. jmedgenet-2019-106672 (2020).
23. Leshno A The APC I1307K allele conveys a significant increased risk for cancer Int. J. Cancer. 2016 138 6 1361 1367 10.1002/ijc.29876 26421687
Leshno, A. et al. The APC I1307K allele conveys a significant increased risk for cancer. Int. J. Cancer. 138(6), 1361–1367 (2016).26421687 10.1002/ijc.29876
24. Paluch-Shimon S Prevention and screening in BRCA mutation carriers and other breast/ovarian hereditary cancer syndromes: ESMO Clinical Practice Guidelines for cancer prevention and screening Ann. Oncol. 2016 27 suppl 5 v103 v110 10.1093/annonc/mdw327 27664246
Paluch-Shimon, S. et al. Prevention and screening in BRCA mutation carriers and other breast/ovarian hereditary cancer syndromes: ESMO Clinical Practice Guidelines for cancer prevention and screening. Ann. Oncol. 27(suppl 5), v103–v110 (2016).27664246 10.1093/annonc/mdw327
25. Yen, T. et al. APC-Associated Polyposis Conditions. GeneReviews. Bookshelf ID: NBK1345. 1998 Dec 18. [updated 2022 May 12].
26. Sweet-Cordero EA Biegel JA The genomic landscape of pediatric cancers: Implications for diagnosis and treatment Science. 2019 363 6432 1170 1175 10.1126/science.aaw3535 30872516
Sweet-Cordero, E. A. & Biegel, J. A. The genomic landscape of pediatric cancers: Implications for diagnosis and treatment. Science. 363(6432), 1170–1175 (2019).30872516 10.1126/science.aaw3535
27. Wong M Whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric cancer Nat. Med. 2020 26 11 1742 1753 10.1038/s41591-020-1072-4 33020650
Wong, M. et al. Whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric cancer. Nat. Med. 26(11), 1742–1753 (2020).33020650 10.1038/s41591-020-1072-4
28. Li S Cancer risks associated with BRCA1 and BRCA2 pathogenic variants J. Clin. Oncol. 2022 40 14 1529 1541 10.1200/JCO.21.02112 35077220
Li, S. et al. Cancer risks associated with BRCA1 and BRCA2 pathogenic variants. J. Clin. Oncol. 40(14), 1529–1541 (2022).35077220 10.1200/JCO.21.02112
29. Kim J Frequency of pathogenic germline variants in cancer-susceptibility genes in the childhood cancer survivor study JNCI Cancer Spectr. 2021 5 2 pka007 10.1093/jncics/pkab007
Kim, J. et al. Frequency of pathogenic germline variants in cancer-susceptibility genes in the childhood cancer survivor study. JNCI Cancer Spectr. 5(2), pka007 (2021).10.1093/jncics/pkab007
30. Zhang J Germline mutations in predisposition genes in pediatric cancer N. Engl. J. Med. 2015 373 24 2336 2346 10.1056/NEJMoa1508054 26580448
Zhang, J. et al. Germline mutations in predisposition genes in pediatric cancer. N. Engl. J. Med. 373(24), 2336–2346 (2015).26580448 10.1056/NEJMoa1508054
31. Slade I DICER1 syndrome: Clarifying the diagnosis, clinical features and management implications of a pleiotropic tumour predisposition syndrome J. Med. Genet. 2011 48 4 273 278 10.1136/jmg.2010.083790 21266384
Slade, I. et al. DICER1 syndrome: Clarifying the diagnosis, clinical features and management implications of a pleiotropic tumour predisposition syndrome. J. Med. Genet. 48(4), 273–278 (2011).21266384 10.1136/jmg.2010.083790
32. Lee JC Primary intracranial sarcomas with DICER1 mutation often contain prominent eosinophilic cytoplasmic globules and can occur in the setting of neurofibromatosis type 1 Acta Neuropathol. 2019 137 3 521 525 10.1007/s00401-019-01960-x 30649606
Lee, J. C. et al. Primary intracranial sarcomas with DICER1 mutation often contain prominent eosinophilic cytoplasmic globules and can occur in the setting of neurofibromatosis type 1. Acta Neuropathol. 137(3), 521–525 (2019).30649606 10.1007/s00401-019-01960-x
33. Zhuang S A transcriptional signature detects homologous recombination deficiency in pancreatic cancer at the individual level Mol. Ther. Nucleic Acids. 2021 26 1014 1026 10.1016/j.omtn.2021.10.014 34786207
Zhuang, S. et al. A transcriptional signature detects homologous recombination deficiency in pancreatic cancer at the individual level. Mol. Ther. Nucleic Acids. 26, 1014–1026 (2021).34786207 10.1016/j.omtn.2021.10.014
34. Fang CB Wu HT Zhang ML Liu J Zhang GJ Fanconi anemia pathway: Mechanisms of breast cancer predisposition development and potential therapeutic targets Front. Cell Dev. Biol. 2020 8 160 10.3389/fcell.2020.00160 32300589
Fang, C. B., Wu, H. T., Zhang, M. L., Liu, J. & Zhang, G. J. Fanconi anemia pathway: Mechanisms of breast cancer predisposition development and potential therapeutic targets. Front. Cell Dev. Biol. 8, 160 (2020).32300589 10.3389/fcell.2020.00160
35. Su X Huang J The Fanconi anemia pathway and DNA interstrand cross-link repair Protein Cell. 2011 2 9 704 711 10.1007/s13238-011-1098-y 21948210
Su, X. & Huang, J. The Fanconi anemia pathway and DNA interstrand cross-link repair. Protein Cell. 2(9), 704–711 (2011).21948210 10.1007/s13238-011-1098-y
36. Nepal M Ma C Xie G Jia W Fei P Fanconi Anemia complementation group C protein in metabolic disorders Aging (Albany, NY). 2018 10 6 1506 1522 10.18632/aging.101487 29930218
Nepal, M., Ma, C., Xie, G., Jia, W. & Fei, P. Fanconi Anemia complementation group C protein in metabolic disorders. Aging (Albany, NY). 10(6), 1506–1522 (2018).29930218 10.18632/aging.101487
37. Kulanuwat S Jungtrakoon P Tangjittipokin W Yenchitsomanus PT Plengvidhya N Fanconi anemia complementation group C protection against oxidative stress-induced β-cell apoptosis Mol. Med. Rep. 2018 18 2 2485 2491 29901137
Kulanuwat, S., Jungtrakoon, P., Tangjittipokin, W., Yenchitsomanus, P. T. & Plengvidhya, N. Fanconi anemia complementation group C protection against oxidative stress-induced β-cell apoptosis. Mol. Med. Rep. 18(2), 2485–2491 (2018).29901137
38. Sager RA Fanconi anemia complementation group C (FANCC) gene association with hereditary and sporadic renal tumors (RT) JCO. 2024 42 4_suppl 450 450 10.1200/JCO.2024.42.4_suppl.450
Sager, R. A. et al. Fanconi anemia complementation group C (FANCC) gene association with hereditary and sporadic renal tumors (RT). JCO. 42(4_suppl), 450–450 (2024).10.1200/JCO.2024.42.4_suppl.450
39. Nieuwenhuis MH A nation-wide study comparing sporadic and familial adenomatous polyposis-related desmoid-type fibromatoses Int. J. Cancer. 2011 129 1 256 261 10.1002/ijc.25664 20830713
Nieuwenhuis, M. H. et al. A nation-wide study comparing sporadic and familial adenomatous polyposis-related desmoid-type fibromatoses. Int. J. Cancer. 129(1), 256–261 (2011).20830713 10.1002/ijc.25664
40. Bader AS Hawley BR Wilczynska A Bushell M The roles of RNA in DNA double-strand break repair Br. J. Cancer. 2020 122 5 613 623 10.1038/s41416-019-0624-1 31894141
Bader, A. S., Hawley, B. R., Wilczynska, A. & Bushell, M. The roles of RNA in DNA double-strand break repair. Br. J. Cancer. 122(5), 613–623 (2020).31894141 10.1038/s41416-019-0624-1
41. Lu WT Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair Nat. Commun. 2018 9 1 532 10.1038/s41467-018-02893-x 29416038
Lu, W. T. et al. Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair. Nat. Commun. 9(1), 532 (2018).29416038 10.1038/s41467-018-02893-x
42. Li MM Standards and guidelines for the interpretation and reporting of sequence variants in cancer: A joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists J. Mol. Diagn. 2017 19 1 4 23 10.1016/j.jmoldx.2016.10.002 27993330
Li, M. M. et al. Standards and guidelines for the interpretation and reporting of sequence variants in cancer: A joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J. Mol. Diagn. 19(1), 4–23 (2017).27993330 10.1016/j.jmoldx.2016.10.002
