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Background: Deletion of 1p is associated with poor prognosis in neuroblastoma, however selected 1p-intact patients still experience poor outcomes. Since mutations of 1p genes may mimic the deleterious effects of chromosomal loss, we studied the incidence, spectrum and effects of mutational variants in 1p-intact neuroblastoma., Methods: We characterized the 1p status of 325 neuroblastoma patients, and correlated the mutational status of 1p tumor suppressors and neuroblastoma candidate genes with survival outcomes among 100 1p-intact cases, then performed functional validation of selected novel variants of 1p36 genes identified from our patient cohort., Results: Among patients with adverse disease characteristics, those who additionally had 1p deletion had significantly worse overall survival. Among 100 tumor-normal pairs sequenced, somatic mutations of 1p tumor suppressors KIF1Bβ and CHD5 were most frequent (2%) after ALK and ATRX (8%), and BARD1 (3%). Mutations of neuroblastoma candidate genes were associated with other synchronous mutations and concurrent 11q deletion (P = 0.045). In total, 24 of 38 variants identified were novel and predicted to be deleterious or pathogenic. Functional validation identified novel KIF1Bβ I1355M variant as a gain-of-function mutation with increased expression and tumor suppressive activity, correlating with indolent clinical behavior; another novel variant CHD5 E43Q was a loss-of-function mutation with decreased expression and increased long-term cell viability, corresponding with aggressive disease characteristics., Conclusions: Our study showed that chromosome 1 gene mutations occurred frequently in 1p-intact neuroblastoma, but may not consistently abrogate the function of bonafide 1p tumor suppressors. These findings may augment the evolving model of compounding contributions of 1p gene aberrations toward tumor suppressor inactivation in neuroblastoma., (© 2022. The Author(s).)

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., (© 2020 John Wiley & Sons Ltd.)

Ex vivo evaluation of personalized models can facilitate individualized treatment selection for patients, and advance the discovery of novel therapeutic options. However, for embryonal malignancies, representative primary cultures have been difficult to establish. We developed patient-derived cell cultures (PDCs) from chemo-naïve and post-treatment neuroblastoma tumors in a consistent and efficient manner, and characterized their in vitro growth dynamics, histomorphology, gene expression, and functional chemo-response. From 34 neuroblastoma tumors, 22 engrafted in vitro to generate 31 individual PDC lines, with higher engraftment seen with metastatic tumors. PDCs displayed characteristic immunohistochemical staining patterns of PHOX2B, TH, and GD2 synthase. Concordance of MYCN amplification, 1p and 11q deletion between PDCs and patient tumors was 83.3%, 72.7%, and 80.0% respectively. PDCs displayed a predominantly mesenchymal-type gene expression signature and showed upregulation of pro-angiogenic factors that were similarly enriched in culture medium and paired patient serum samples. When tested with standard-of-care cytotoxics at human C max -equivalent concentrations, MYCN-amplified and non-MYCN-amplified PDCs showed a differential response to cyclophosphamide and topotecan, which mirrored the corresponding patients' responses, and correlated with gene signatures of chemosensitivity. In this translational proof-of-concept study, early-phase neuroblastoma PDCs enriched for the mesenchymal cell subpopulation recapitulated the individual molecular and phenotypic profile of patient tumors, and highlighted their potential as a platform for individualized ex vivo drug-response testing., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objective: To investigate the clinical and pathological features of pediatric NTRK-rearranged tumors. Methods: Four NTRK-rearranged soft tissue tumors and one renal tumor at Shanghai Children's Medical Center, Shanghai Jiaotong University and Singapore KK Women's and Children's Hospital from January 2017 to September 2019 were identified. Pan-TRK immunohistochemistry, and the ALK and ETV6 gene break-apart fluorescence in situ hybridizations (FISH) were performed. NTRK gene rearrangement was detected using sequencing-based methods. Results: There were 3 males and 2 females in this study. The patients were between 3 months and 13 years of age. Histologically, the tumors were infiltrative spindle cell tumors with variable accompanying inflammatory cells. Immunohistochemistry showed positive reactivity for pan-TRK in all tumors, with nuclear staining for NTRK3 fusion, and cytoplasmic staining for NTRK1 fusion. The molecular testing revealed NTRK gene fusions (one each of TPM3-NTRK1, ETV6-NTRK3 and DCTN1-NTRK1, and two cases of LMNA-NTRK1). Two patients were receiving larotrectinib. The others were are well without disease, with follow-up durations of 9 to 29 months. Conclusions: NTRK-rearranged mesenchymal tumors from soft tissue sites and kidney are identified. A novel DCTN1-NTRK1 fusion is described. Pan-TRK immunohistochemistry is useful for diagnosis. NTRK-targeted therapy may be an option for unresectable, recurrent or metastatic cases.

Objective: To study clinical pathological characteristics, immunohistochemical, molecular genetical changes and prognosis in pediatric eosinophilic solid and cystic renal cell carcinoma (ESC RCC) with TSC2 gene mutations. Methods: The tissue samples were collected from two pediatric ESC RCC patients between 2017 and 2018. The tissues were subjected to histological examination and immunohistochemistry using EnVision system. The TFE3, TFEB gene rearrangements were tested using FISH and molecular genetic study. The paraffin sections were used for DNA extraction, PCR amplification and NGS sequencing. Results: The two patients with ESC RCC were both male, aged at 9 years and 8 months, and 13 years, respectively. The tumors were from the right kidney, 5 cm and 7 cm in size, respectively, with solid and cystic changes in cross section, and grey-reddish or grey-whitish fish meat appearance. Microscopic observation revealed the tumors had fibrous capsules, which were infiltrated by the tumor cells. The tumor cells were diffusely distributed, round-shaped, or polygon-shaped, and had voluminous cytoplasm, eosinophilic cytoplasm, various sizes of vacuoles and clear cell-like appearance. There were papillary structures in some areas, with visible fiber septa. The nuclei were round and vesicular, with multi-nucleated cells and megakaryocytes. The mitoses were not seen. A few cystic structures were visible in different sizes, and capsule walls were covered with a single layer of spike-like tumor cells. Thick-walled blood vessels were seen in the stroma, with focal lymphocytic infiltration, eosinophilic necrosis, calcifications and cholesterol crystals. Immunohistochemistry of the tumor cells was positive for PAX8 (diffuse), CK20 (focal), CKpan (focal), CK10 (1 focal, 1 diffuse), INI1, vimentin, CD68, and Ki-67 (5%~10%); the tumor cells were negative for HMB45, S-100, Melan A, p53, desmin, TFE3, CK7, CK19, EMA, CD56, CgA, Syn, CD30, CD117, WT1 and SMA. Molecular genetic study showed that TFE3 and TFEB gene rearrangements were not detected by FISH. NGS sequencing showed TSC2 p.Lys574Ter (0.198) was found in patient one and TSC2 p.Arg406Ter (0.355) in patient two. Conclusions: ESC RCC in children is a rare disease, and can be misdiagnosed easily. It has unique pathological characteristics, and immunohistochemical, molecular and genetic changes. The prognosis is relatively good.