RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Imaging characterization of paediatric tumours with the neurotrophic tyrosine receptor kinase fusion transcript.

OBJECTIVES: The neurotrophic tyrosine receptor kinase (NTRK) fusion transcript (FT) is a major genetic landmark of infantile fibrosarcoma (IFS) and cellular congenital mesoblastic nephroma (cCMN) but is also described in other tumours. The recent availability of NTRK-targeted drugs enhances the need for better identification. We aimed to describe the anatomic locations and imaging features of tumours with NTRK-FT in children. CASE SERIES: Imaging characteristics of NTRK-FT tumours of 41 children (median age: 4 months; 63% <1 year old; range: 0-188) managed between 2001 and 2019 were retrospectively analysed. The tumours were located in the soft tissues (n = 24, including 19 IFS), kidneys (n = 9, including 8 cCMN), central nervous system (CNS) (n = 5), lung (n = 2), and bone (n = 1). The tumours were frequently deep-located (93%) and heterogeneous (71%) with necrotic (53%) or haemorrhagic components (29%). Although inconstant, enlarged intratumoural vessels were a recurrent finding (70%) with an irregular distribution (63%) in the most frequent anatomical locations. CONCLUSION: Paediatric NTRK-FT tumours mainly occur in infants with very variable histotypes and locations. Rich and irregular intra-tumoural vascularization are recurrent findings. ADVANCES IN KNOWLEDGE: Apart from IFS of soft tissues and cCMN of the kidneys, others NTRK-FT tumours locations have to be known, as CNS tumours. Better knowledge of the imaging characteristics may help guide the pathological and biological identification.

Hypermethylation of RASSF1A gene in pediatric rhabdoid tumor of the kidney and clear cell sarcoma of the kidney.

BACKGROUND: Among pediatric renal tumors, rhabdoid tumor of the kidney (RTK) and clear cell sarcoma of the kidney (CCSK) are rare and associated with an unfavorable prognosis, while congenital mesoblastic nephroma (CMN) is associated with a good prognosis. Methylation of the Ras association domain-containing protein 1 isoform A (RASSF1A) promoter has been reported to correlate with a poor prognosis in patients with Wilms tumors, while its methylation status is unclear in other types of pediatric renal tumors. METHOD: DNA methylation of the RASSF1A promoter in several pediatric renal tumors was analyzed with pyrosequencing. In order to clarify the correlation between expression of RASSF1A and DNA methylation of its promoter, the RTK cell line was treated with 5-Aza-2'-deoxycytidine (5-Aza-dC). RASSF1A was overexpressed in the RTK cell line to evaluate its functional effects. RESULTS: Quantitative methylation analysis demonstrated hypermethylation in the RASSF1A promoter region in RTK and CCSK, but not CMN. The 5-Aza-dC treatment induced demethylation of the RASSF1A promoter as well as increased RASSF1A mRNA expression. The transduction of RASSF1A has an effect on the suppression of viability and proliferation of RTK cells. CONCLUSION: DNA methylation-mediated deficiency of RASSF1A might be involved in the development and aggressiveness of some pediatric renal tumors and correlated with a poor prognosis.

EGFR internal tandem duplications in fusion-negative congenital and neonatal spindle cell tumors.

Next-generation sequencing (NGS) assays can sensitively detect somatic variation, and increasingly can enable the identification of complex structural rearrangements. A subset of infantile spindle cell sarcomas, particularly congenital mesoblastic nephromas with classic or mixed histology, have structural rearrangement in the form of internal tandem duplications (ITD) involving EGFR. We performed prospective analysis to identify EGFR ITD through clinical or research studies, as well as retrospective analysis to quantify the frequency of EGFR ITD in pediatric sarcomas. Within our institution, three tumors with EGFR ITD were prospectively identified, all occurring in patients less than 1 year of age at diagnosis, including two renal tumors and one mediastinal soft tissue tumor. These three cases exhibited both cellular and mixed cellular and classic histology. All patients had no evidence of disease progression off therapy, despite incomplete resection. To extend our analysis and quantify the frequency of EGFR ITD in pediatric sarcomas, we retrospectively analyzed a cohort of tumors (n = 90) that were previously negative for clinical RT-PCR-based fusion testing. We identified EGFR ITD in three analyzed cases, all in patients less than 1 year of age (n = 18; 3/18, 17%). Here we expand the spectrum of tumors with EGFR ITD to congenital soft tissue tumors and report an unusual example of an EGFR ITD in a tumor with cellular congenital mesoblastic nephroma histology. We also highlight the importance of appropriate test selection and bioinformatic analysis for identification of this genomic alteration that is unexpectedly common in congenital and infantile spindle cell tumors.

Infantile fibrosarcoma with an EGFR kinase domain duplication: Underlining a close relationship with congenital mesoblastic nephroma and highlighting a similar morphological spectrum.

Infantile fibrosarcoma (IFS) and congenital mesoblastic nephroma (CMN) are locally aggressive tumors primarily occurring in infants. Both IFS and the cellular subtype of CMN show overlapping morphological features and an ETV6-NTRK3 fusion, suggesting a close relationship. An activating alteration of EGFR, based on an EGFR kinase domain duplication (KDD), occurs in a subset of CMNs lacking an NTRK3 rearrangement, especially in the classic and mixed type. So far no EGFR-KDDs have been detected in IFS. We describe four pediatric tumors at the extremities (leg, n = 2; foot and arm n = 1) with histological features of IFS/CMN. Two cases showed classic IFS morphology while two were similar to classic/mixed type CMN. In all cases, an EGFR-KDD was identified without detection of a fusion gene. There were no abnormalities of the kidneys in any of the patients. This is the first description of IFS with an EGFR-KDD as driver mutation, supporting that IFS and CMN are similar lesions with the same morphological and genetic spectrum. Pathologists should be aware of the more fibrous variant of IFS, similar to classic/mixed type CMN. Molecular analyses are crucial to treat these lesions adequately, especially with regard to the administration of tyrosine kinase inhibitors.

Mesenchymal neoplasms with NTRK and other kinase gene alterations.

Kinase alterations are increasingly recognised as oncogenic drivers in mesenchymal tumours. Infantile fibrosarcoma and the related renal tumour, congenital mesoblastic nephroma, were among the first solid tumours shown to harbour recurrent tyrosine kinase fusions, with the canonical ETV6::NTRK3 fusion identified more than 20 years ago. Although targeted testing has long been used in diagnosis, the advent of more robust sequencing techniques has driven the discovery of kinase alterations in an array of mesenchymal tumours. As our ability to identify these genetic alterations has improved, as has our recognition and understanding of the tumours that harbour these alterations. Specifically, this study will focus upon mesenchymal tumours harbouring NTRK or other kinase alterations, including tumours with an infantile fibrosarcoma-like appearance, spindle cell tumours resembling lipofibromatosis or peripheral nerve sheath tumours and those occurring in adults with a fibrosarcoma-like appearance. As publications describing the histology of these tumours increase so, too, do the variety kinase alterations reported, now including NTRK1/2/3, RET, MET, RAF1, BRAF, ALK, EGFR and ABL1 fusions or alterations. To date, these tumours appear locally aggressive and rarely metastatic, without a clear link between traditional features used in histological grading (e.g. mitotic activity, necrosis) and outcome. However, most of these tumours are amenable to new targeted therapies, making their recognition of both diagnostic and therapeutic import. The goal of this study is to review the clinicopathological features of tumours with NTRK and other tyrosine kinase alterations, discuss the most common differential diagnoses and provide recommendations for molecular confirmation with associated treatment implications.

An unusual fusion gene EML4-ALK in a patient with congenital mesoblastic nephroma.

Congenital mesoblastic nephroma (CMN), the most common renal tumor of infancy, is a mesenchymal neoplasm histologically classified into classic, cellular, or mixed types. Most cellular CMNs harbor a characteristic ETV6-NTRK3 fusion. Here, we report an unusual congenital mesoblastic nephroma presenting in a newborn boy with a novel EML4-ALK gene fusion revealed by Anchored Multiplex RNA Sequencing Assay. The EML4-ALK gene fusion expands the genetic spectrum implicated in the pathogenesis of congenital mesoblastic nephroma, with yet another example of kinase oncogenic activation through chromosomal rearrangement. The methylation profile of the tumor corresponds with infantile fibrosarcoma showing the biological similarity of these two entities.

Expanding Phenotype of Schimke Immuno-Osseous Dysplasia: Congenital Anomalies of the Kidneys and of the Urinary Tract and Alteration of NK Cells.

Schimke immuno-osseous dysplasia (SIOD) is a rare multisystemic disorder with a variable clinical expressivity caused by biallelic variants in SMARCAL1. A phenotype-genotype correlation has been attempted and variable expressivity of biallelic SMARCAL1 variants may be associated with environmental and genetic disturbances of gene expression. We describe two siblings born from consanguineous parents with a diagnosis of SIOD revealed by whole exome sequencing (WES). Results: A homozygous missense variant in the SMARCAL1 gene (c.1682G>A; p.Arg561His) was identified in both patients. Despite carrying the same variant, the two patients showed substantial renal and immunological phenotypic differences. We describe features not previously associated with SIOD-both patients had congenital anomalies of the kidneys and of the urinary tract and one of them succumbed to a classical type congenital mesoblastic nephroma. We performed an extensive characterization of the immunophenotype showing combined immunodeficiency characterized by a profound lymphopenia, lack of thymic output, defective IL-7Rα expression, and disturbed B plasma cells differentiation and immunoglobulin production in addition to an altered NK-cell phenotype and function. Conclusions: Overall, our results contribute to extending the phenotypic spectrum of features associated with SMARCAL1 mutations and to better characterizing the underlying immunologic disorder with critical implications for therapeutic and management strategies.

Pediatric Renal Tumors: Updates in the Molecular Era.

Molecular characterization has led to advances in the understanding of pediatric renal tumors, including the association of pediatric cystic nephromas with DICER1 tumor syndrome, the metanephric family of tumors with somatic BRAF mutations, the characterization of ETV6-NTRK3-negative congenital mesoblastic nephromas, the expanded spectrum of gene fusions in translocation renal cell carcinoma, the relationship of clear cell sarcoma of the kidney with other BCOR-altered tumors, and the pathways affected by SMARCB1 alterations in rhabdoid tumors of the kidney. These advances have implications for diagnosis, classification, and treatment of pediatric renal tumors.

Congenital mesoblastic nephroma is characterised by kinase mutations including EGFR internal tandem duplications, the ETV6-NTRK3 fusion, and the rare KLHL7-BRAF fusion.

AIMS: Congenital mesoblastic nephroma (CMN) is histologically classified into classic, cellular and mixed subtypes. The aims of this study were to characterise the clinical, pathological and molecular features of a series of CMNs, and to determine the utility of pan-Trk and epidermal growth factor receptor (EGFR) immunohistochemistry as surrogate markers for NTRK gene fusions and EGFR internal tandem duplications (ITDs). METHODS AND RESULTS: Twenty-two archival CMN cases (12 classic, five cellular, and five mixed) were tested for the ETV6-NTRK3 fusion and EGFR ITD transcripts by the use of reverse transcriptase polymerase chain reaction (PCR), and next-generation sequencing-based anchored multiplex PCR. All 12 classic CMNs had EGFR ITD. Of the five cellular CMNs, four had the ETV6-NTRK3 fusion and one had the KLHL7-BRAF fusion. Of the five mixed CMNs, four had EGFR ITD, and one had the ETV6-NTRK3 fusion. Pan-Trk immunoreactivity was 100% sensitive and 94.1% specific for the presence of NTRK rearrangement. However, EGFR staining was only 62.5% sensitive and 33.3% specific for EGFR ITD. CONCLUSIONS: EGFR ITD is a consistent genetic event in classic CMN. A majority of cellular CMNs have the ETV6-NTRK3 fusion. Rare cellular CMNs may harbour non-canonical mutations such as the KLHL7-BRAF fusion, which was found in one case. Mixed CMNs may have either EGFR ITD or the ETV6-NTRK3 fusion. Pan-Trk immunohistochemistry is a sensitive, albeit not perfectly specific, marker for NTRK rearrangement. EGFR immunohistochemistry is not helpful as a marker of EGFR ITD.

An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.

[Rare childhood kidney tumors]. / Seltene kindliche Nierentumoren.

Pediatric kidney tumors are rare and account for about 6% of all childhood malignancies. By far the most common tumors are nephroblastomas. This review presents rare childhood renal tumors. Mesoblastic nephroma, as tumors of the low risk group, as well as the clear-cell sarcomas of the kidney and malignant rhabdoid tumors, as tumors of the high-risk group, and the so-called anaplastic sarcomas of the kidney will be discussed.Due to the significantly divergent therapy, a correct diagnosis is important. Due to the often overlapping morphology, pathologic diagnosis is often difficult. In addition to the typical morphologic features, the specific immunohistochemical aspects as well as the known molecular changes will be presented.

Dramatic bone remodeling following larotrectinib administration for bone metastasis in a patient with TRK fusion congenital mesoblastic nephroma.

Mesoblastic nephroma is the most frequent renal tumor in newborns and young infants, and the cellular type is characterized by an ETV6-NTRK fusion, which constitutively activates the tropomyosin-related kinase (TRK) signaling pathway. Larotrectinib is a highly selective TRK inhibitor with activity in adult and pediatric patients who have TRK fusions. We present a rare case of a patient with mesoblastic nephroma metastatic to bone who had a dramatic response to larotrectinib.

Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants.

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors.

Pan-Trk Immunohistochemistry Identifies NTRK Rearrangements in Pediatric Mesenchymal Tumors.

Activating neurotrophic receptor kinase (NTRK) fusions define certain pediatric mesenchymal tumors, including infantile fibrosarcoma and cellular mesoblastic nephroma. Traditionally, molecular confirmation of these fusions has included either fluorescent in situ hybridization for ETV6 rearrangements or reverse-transcriptase polymerase chain reaction for the classic ETV6-NTRK3 fusion. However, these methods overlook variant NTRK rearrangements, which are increasingly appreciated as recurrent events in a subset of pediatric mesenchymal tumors. New therapeutic agents successfully target these fusions and may prevent morbid surgeries in very young children, making recognition of tumors harboring NTRK rearrangements of increasing importance. We evaluated the performance of immunohistochemical (IHC) staining using pan-Trk and TrkA antibodies in 79 pediatric mesenchymal tumors. Negative controls included pediatric mesenchymal tumors not harboring (n=28) or not expected to harbor (n=22) NTRK fusions. NTRK rearrangements were detected predominantly by DNA-based next-generation sequencing assays, specifically UW OncoPlex and UCSF500 Cancer Gene Panel. Pan-Trk IHC (EPR17341) was 97% sensitive and 98% specific for the presence of an NTRK rearrangement, and TrkA IHC (EP1058Y) was 100% sensitive and 63% specific for the presence of an NTRK rearrangement. Tumors with NTRK1 or NTRK2 rearrangements showed cytoplasmic staining, whereas tumors with NTRK3 rearrangements showed nuclear +/- cytoplasmic staining. We conclude that pan-Trk IHC is a highly sensitive and specific marker for NTRK rearrangements in pediatric mesenchymal tumors.

Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy.

Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6-NTRK3 gene fusion. However, a number of case reports have identified variant fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4-NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6-NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4-NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4-NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4-NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients.

ETV6-NTRK3 in congenital mesoblastic nephroma: A report of the SIOP/GPOH nephroblastoma study.

BACKGROUND: Congenital mesoblastic nephroma (MN) is a rare pediatric renal tumor representing approximately 5% of all pediatric renal tumors. Three different types of MN are distinguished histologically: classical, cellular, and mixed. A frequent genetic alteration is the translocation t(12;15) resulting in a fusion of the ETV6 gene on 12p13 and the NTRK3 gene on 15p15 that occurs almost exclusively in cellular MN. The aim of this study was to determine translocation status of a large cohort of MN with respect to tumor subtype and outcome. PROCEDURE: In total, clinical data from 111 patients were available. Sixty-seven tumors were classical MN (51%), 29 cellular MN (31%), and 15 were mixed MN (18%). From these 111 cases, 79 were analyzed by FISH and RT-PCR. RESULTS: All classical and mixed MN were translocation negative. Seventeen out of 29 (58%) cellular MN harbored the ETV6-NTRK3 translocation. Five-year relapse-free survival (RFS) and overall survival (OS) were 93.2% and 96.8% for the complete cohort. All seven relapses occurred in translocation negative tumors. Five-year RFS was significantly inferior for cellular and mixed MN compared to classic MN (89%, 80%, and 98%), whereas 5-year OS was similar (93%, 96%, and 98%). Within the group of cellular MN, patients having translocation-positive tumors had a significantly superior RFS (5-year RFS: 100% vs. 73%). CONCLUSION: The majority of cellular MNs harbor the ETV6-NTKR3 gene fusion, whereas all classic- and mixed-type MNs were translocation negative. Within the cellular subgroup, patients having translocation-positive tumors had a significantly superior RFS.

[Solid pediatric tumors : A brief survey of the rarity cabinet]. / Solide Kindertumoren : Ein Streifzug durch das Raritätenkabinett.

Solid tumors in childhood are extremely rare entities, which are usually treated in specialized centers. Diagnosis and therapy are carried out according to a joint European protocol, whereby the pathological evaluation and therapy are carried out according to international guidelines. For the correct diagnosis and/or therapy of most tumors, analysis of specific genetic changes is mandatory; therefore, tumors have to be adequately sampled for parallel genetic analysis during the pathological work-up. A second opinion reference of the histopathological assessment is part of the international guidelines. Neuroblastomas, congenital mesoblastic nephromas and rhabdoid tumors are examples of solid tumors in childhood that are not restricted to one organ and occur exclusively during childhood.

Identification of somatic and germ-line DICER1 mutations in pleuropulmonary blastoma, cystic nephroma and rhabdomyosarcoma tumors within a DICER1 syndrome pedigree.

BACKGROUND: DICER1 syndrome is a pediatric cancer predisposition condition causing a variety of tumor types in children and young adults. In this report we studied a family with two relatives presenting a variety of neoplastic conditions at childhood. METHODS: Germ-line mutation screening of the complete coding region of the DICER1 gene in genomic DNA from the proband was performed. The presence of somatic DICER1 mutation and further alterations in driver genes was investigated in genomic DNA obtained from available tumor samples. RESULTS: A nonsense germ-line mutation in DICER1 causing a truncated protein at the IIIb domain level was identified segregating within a family including two affected relatives who developed in one case cystic nephroma and pleuropulmonary blastoma, and rhabdomyosarcoma and multinodular goiter in the other. Additional in trans DICER1 missense somatic mutations in the IIIb DICER1 domain were found both in the cystic nephroma and in the rhabdomyosarcoma, suggesting that neoplasms in this family might arise from the unusual two-hit mechanism for DICER-derived tumorigenesis in which after the presence of a truncated constitutive protein, a neomorphic DICER1 activity is somatically adquired. Additional genetic alterations, such as TP53 mutations, were identified in the rhabdomyosarcoma. CONCLUSIONS: Besides DICER1 loss of standard activity, oncogenic cooperation of other genes, as mutated TP53, may involve developing higher grade tumors within this syndrome. Given the broad clinical spectrum that may arise, genetic counseling and close surveillance must be offered to all family members at risk of DICER1 syndrome.