SUMMARYFertilization in flowering plants involves two sperm cells and two female gametes, the egg cell and the central cell, progenitors of the embryo and the endosperm, respectively. The mechanisms triggering zygotic development are unknown and whether both parental genomes are required for zygotic development is unclear. In Arabidopsis, previous studies reported that loss-of-function mutations in CYCLIN DEPENDENT KINASE A1 (CDKA;1) impedes cell cycle progression in the pollen leading to the production of a single sperm cell. Here, we report that a significant proportion of single cdka;1 pollen delivers two sperm cells, leading to a new assessment of the cdka;1 phenotype. We performed fertilization of wild-type ovules with cdka;1 mutant sperm cells and monitored in vivo the fusion of the male and female nuclei using fluorescent markers. When a single cdka;1 sperm was delivered, either female gamete could be fertilized leading to similar proportions of seeds containing either a single endosperm or a single embryo. When two cdka;1 sperm cells were released, they fused to each female gamete. Embryogenesis was initiated but the fusion between the nuclei of the sperm cell and the central cell failed. The failure of karyogamy in the central cell prevented incorporation of the paternal genome, impaired endosperm development and caused seed abortion. Our results thus support that the paternal genome plays an essential role during early seed development. However, sperm entry was sufficient to trigger central cell mitotic division, suggesting the existence of signaling events associated with sperm cell fusion with female gametes.
The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.
Clear cell sarcoma of the kidney is the second most common primary renal malignancy in childhood. It is histologically diverse, making accurate diagnosis challenging in some cases. Recent molecular studies have uncovered BCOR exon 15 internal tandem duplications in most cases, and YWHAE-NUTM2 fusion in a few cases, with the remaining cases having other genetic mutations, including BCOR-CCNB3 fusion and EGFR mutations. Although clear cell sarcoma of the kidney has no specific immunophenotype, several markers including cyclin D1, nerve growth factor receptor, and BCOR (BCL6 corepressor) have emerged as potential diagnostic aides. This review provides a concise account of recent advances in our understanding of clear cell sarcoma of the kidney to serve as a practical update for the practicing pathologist.
Pathological diagnosis of clear cell sarcoma of the kidney (CCSK) is challenging as it resembles blastemal Wilms tumor (WT) and other pediatric sarcomas, and does not have any distinctive immunophenotype. The YWHAE-FAM22 translocation t(10;17)(q22;p13) has been reported in a subset of CCSK. This translocation also occurs in high-grade endometrial sarcoma, in which it is associated with cyclin D1 overexpression. Hence we seek to determine YWHAE-FAM22 translocation status and cyclin D1 immunoreactivity in a series of local CCSK cases. Of 8 CCSK cases from 7 patients identified, no CCSK had the YWHAE-FAM22 fusion transcript by reverse transcriptase-polymerase chain reaction. Novel karyotypes were identified for 2 cases: 1 had t(2;13)(q13;q22) and the other t(3:17)(q29;p11.2). Excluding a case with poor tissue section antigenicity, 7 of 7 CCSKs (100%) showed diffuse and strong nuclear cyclin D1 staining. Cyclin D1 immunohistochemistry was also performed on tissue microarrays of other pediatric renal tumors: blastemal areas of 18 WT cases were negative; 6 rhabdoid tumors and 1 metanephric adenoma showed patchy and weak staining; 3 mesoblastic nephromas and 18 of 29 neuroblastomas had positive staining. Cyclin D1 immunohistochemistry helps distinguish CCSK from blastemal WT and metanephric adenoma and rhabdoid tumors, but not from neuroblastomas and mesoblastic nephromas. Cyclin D1 overexpression in CCSK is not contingent on YWHAE-FAM22 translocation, and cyclin D1 inhibition may potentially be explored as a targeted therapeutic strategy in CCSK.
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.
Clear cell sarcoma of the kidney (CCSK) and primitive myxoid mesenchymal tumour of infancy (PMMTI) are paediatric sarcomas that most commonly harbour internal tandem duplications (ITDs) of exon 15 of the BCOR gene, in the range of 87–114 base pairs (bp). Some cases, instead, have BCOR‐CCNB3 or YWHAE‐NUTM2 gene fusions. About 10% of cases lack any of these genetic alterations when tested by standard methods. Two cases of CCSK and one PMMTI lacking the aforementioned mutations were analysed using Archer FusionPlex technology. Two related BCOR exon 15 RNA transcripts with ITDs of lengths 388 and 96 bp were detected in each case; only the 388 bp transcript was identified when genomic DNA was sequenced. In silico analysis of this transcript revealed acceptor and donor splice sites indicating that, at the RNA level, the 388‐bp transcript was likely spliced to form the 96‐bp transcript. The results were confirmed by Sanger sequencing using primers targeting the ITD breakpoint. This novel and unusually long ITD segment is difficult to identify by DNA sequencing using typical primer design strategies flanking entire duplicated segments because it exceeds the typical read lengths of most sequencing platforms as well as the usual fragment lengths obtained from formalin‐fixed paraffin‐embedded material. As diagnosis of CCSK and PMMTI may be challenging by morphology and immunohistochemistry alone, it is important to identify mutations in these cases. Knowledge of this novel BCOR ITD is important in relation to primer design for detection by sequencing, and using RNA versus DNA for sequencing.
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