Single-cell multiomics reveals the interplay of clonal evolution and cellular plasticity in hepatoblastoma.

Hepatoblastomas (HB) display heterogeneous cellular phenotypes that influence the clinical outcome, but the underlying mechanisms are poorly understood. Here, we use a single-cell multiomic strategy to unravel the molecular determinants of this plasticity. We identify a continuum of HB cell states between hepatocytic (scH), liver progenitor (scLP) and mesenchymal (scM) differentiation poles, with an intermediate scH/LP population bordering scLP and scH areas in spatial transcriptomics. Chromatin accessibility landscapes reveal the gene regulatory networks of each differentiation pole, and the sequence of transcription factor activations underlying cell state transitions. Single-cell mapping of somatic alterations reveals the clonal architecture of each tumor, showing that each genetic subclone displays its own range of cellular plasticity across differentiation states. The most scLP subclones, overexpressing stem cell and DNA repair genes, proliferate faster after neo-adjuvant chemotherapy. These results highlight how the interplay of clonal evolution and epigenetic plasticity shapes the potential of HB subclones to respond to chemotherapy.

The RELIVE consortium for relapsed or refractory pediatric hepatoblastoma and hepatocellular carcinoma: a scoping review of the problem and a proposed solution.

Liver tumors account for approximately 2% of all pediatric malignancies. Children with advanced stages of hepatoblastoma (HB) are cured only 50-70% of the time while children with advanced hepatocellular carcinoma (HCC) have a <20% 5-year overall survival. This scoping review was performed to highlight the paucity of rigorous, reliable data guiding the management of relapsed pediatric HB or HCC. When these patients are enrolled on prospective trials, the trials are often histology-agnostic, exclude patients less than a year of age, lack a liquid formulary of the drug under study, exclude recipients of a solid organ transplant, and enroll only 1-2 patients limiting the ability to deduce efficacious regimens for current use or future study. We highlight the creation of a global pediatric consortium intended to source retrospective relapse data from over 100 institutions spanning 4 continents. The data collected from this effort will inform future relapse trials.

Second Malignant Neoplasms Following Treatment for Hepatoblastoma: An International Report and Review of the Literature.

Treatment intensification has improved survival in patients with hepatoblastoma (HB); however, these treatments are associated with an increased risk of late effects, including second malignant neoplasms (SMNs). Data is limited regarding SMNs following HB treatment. Cases of SMNs following treatment for HB reported in the literature and from personal communication were analyzed to further assess this late effect. Thirty-eight patients were identified. The median age at diagnosis of HB was 16 months (range: 3 to 168 mo). All patients had received a platinum agent, and almost all had anthracycline exposure. The SMNs reported were hematopoietic malignancies (n=19), solid tumors (n=12), and post-transplant lymphoproliferative disorder (n=7). Of the 36 patients with outcome data, 19 survived. SMNs following HB treatment were primarily seen in patients with chemotherapy exposure, a history of liver transplantation, hereditary tumor predisposition syndromes, and/or a history of radiation treatment. Hematopoietic malignancies were the most common SMN reported in this cohort and were diagnosed earlier than other SMNs. Prospective collection of data through a companion late effects study or international registry could be used to further evaluate the rates and risks of SMNs as well as tumor predisposition syndromes in patients treated for HB.

Mutational signature, cancer driver genes mutations and transcriptomic subgroups predict hepatoblastoma survival.

BACKGROUND: Hepatoblastoma is the most frequent pediatric liver cancer. The current treatments lead to 80% of survival rate at 5 years. In this study, we evaluated the clinical relevance of molecular features to identify patients at risk of chemoresistance, relapse and death of disease. METHODS: All the clinical data of 86 children with hepatoblastoma were retrospectively collected. Pathological slides were reviewed, tumor DNA sequencing (by whole exome, whole genome or target) and transcriptomic profiling with RNAseq or 300-genes panel were performed. Associations between the clinical, pathological, mutational and transcriptomic data were investigated. RESULTS: High-risk patients represented 44% of our series and the median age at diagnosis was 21.9 months (range: 0-208). Alterations of the WNT/ß-catenin pathway and of the 11p15.5 imprinted locus were identified in 98% and 74% of the tumors, respectively. Other cancer driver genes mutations were only found in less than 11% of tumors. After neoadjuvant chemotherapy, disease-specific survival and poor response to neoadjuvant chemotherapy were associated with 'Liver Progenitor' (p = 0.00049, p < 0.0001) and 'Immune Cold' (p = 0.0011, p < 0.0001) transcriptomic tumor subtypes, SBS35 cisplatin mutational signature (p = 0.018, p = 0.001), mutations in rare cancer driver genes (p = 0.0039, p = 0.0017) and embryonal predominant histological type (p = 0.0013, p = 0.0077), respectively. Integration of the clinical and molecular features revealed a cluster of molecular markers associated with resistance to chemotherapy and survival, enlightening transcriptomic 'Immune Cold' and Liver Progenitor' as a predictor of survival independent of the clinical features. CONCLUSIONS: Response to neoadjuvant chemotherapy and survival in children treated for hepatoblastoma are associated with genomic and pathological features independently of the clinical features.

Achaete-scute family bHLH transcription factor 2 activation promotes hepatoblastoma progression.

Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.

Comprehensive genomic profiling testing in Japanese castration-resistant prostate cancer patients: results of a single-center retrospective cohort study.

OBJECTIVE: Comprehensive genomic profiling testing using a hybrid-capture next-generation sequencing is commonly used in clinical practice to employ precision medicine in cancer treatment worldwide. In this study, we aimed to analyze the profiles obtained using comprehensive genomic profiling testing that was performed in Japanese castration-resistant prostate cancer patients and to discuss the genetic findings in a real-world setting. METHODS: A total of 60 cases and 57 castration-resistant prostate cancer patients underwent comprehensive genomic profiling testing between 1 January 2021 and 31 December 2022. Four types of comprehensive genomic profiling testing were selected, and clinically significant cancer-specific gene alterations were identified. RESULTS: The median age of patients was 74 years, and the median prostate-specific antigen value at the time of submission was 18.6 ng/ml. Fifty-seven (95%) of 60 cases were metastatic castration-resistant prostate cancers, and 3 cases (5%) were non-metastatic. Among all genetic alterations, androgen-receptor alteration was the most frequently detected in 17 cases (28.3%), followed by 15 cases of TP53 (25.0%), 14 cases of CDK12 (23.3%), 10 cases of phosphatase and tensin homolog (16.7%) and 9 cases of ATM (15.0%) mutations. A total of 13 patients (21.7%) received systemic therapy according to the comprehensive genomic profiling testing results. Overall, the survival rate was significantly greater in the group treated through systemic therapy based on comprehensive genomic profiling testing compared with the group without new therapeutic treatment (P = 0.041). CONCLUSIONS: Comprehensive genomic profiling testing is recommended in castration-resistant prostate cancer patients identified as resistant to standard therapy as this can provide a new therapeutic option.

Increased plasma miR-370-3p expression in poor-outcome patients with pancreatic ductal adenocarcinoma.

OBJECTIVE: To determine whether circulating microRNAs (miRNAs) can be used as prognostic biomarkers for pancreatic ductal adenocarcinoma (PDAC). METHODS: Patients with PDAC (N = 120) who underwent surgical resection at Hiroshima University Hospital between November 2006 and January 2020 were enrolled in this study and grouped based on their overall survival (OS) into two groups: favorable prognosis group (F group; OS ≥ 18 months) and unfavorable prognosis group (U group; OS < 18 months). Blood plasma samples were collected prior to surgery. To identify candidate prognostic miRNAs, next-generation sequencing (NGS) analysis was used to evaluate the expression levels of miRNAs in seven of the plasma samples. Using quantitative real-time PCR (qRT-PCR), the expression levels of the selected miRNAs were determined in the remaining 113 patient plasma samples, and the relationship between miRNA expression and survival was statistically evaluated. RESULTS: NGS analysis and qRT-PCR revealed significantly upregulated plasma miR-370-3p expression in the U group compared to that in the F group (p = 0.028 and p = 0.005, respectively). Moreover, miR-370-3p expression and lymph node metastasis showed a statistically significant association (p = 0.028). In a multivariate analysis of OS and recurrence-free survival (RFS), the upregulation of miR-370-3p expression in plasma was identified as an independent risk factor for poor OS (HR2.13, p = 0.004) and RFS (HR1.84, p = 0.015). CONCLUSIONS: Plasma miR-370-3p expression upregulation correlates with poor prognosis in patients with PDAC.

Circulating Tumor Cells and Tumor Progression, Metastasis, and Poor Prognosis in Patients With Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor in children, and the overall survival rate of patients in the high-risk group is less than 50%. Circulating tumor cells have recently been found to be a potential source of tumor progression, metastasis, and poor prognosis in patients with cancer. There is a dearth of data on the malignant potential of circulating tumor cells in neuroblastoma. Therefore, in this article, we briefly review the methods of quantifying circulating neuroblastoma cells and the relationship between the presence of many circulating tumor cells and prognosis for patients with neuroblastoma. The relationship between higher numbers of circulating neuroblastoma tumor cells and poor prognosis was outlined. However, the data in most studies represented only the tumor burden, and there is no direct evidence that circulating tumor cells are the source of tumor progression and metastasis in patients with neuroblastoma. A comprehensive genetic analysis of circulating tumor cells, including single-cell sequencing and functional assays using ex vivo cell culture and xenografts, may provide insights into the malignant nature of neuroblastoma.

Consensus classification of pediatric hepatocellular tumors: A report from the Children's Hepatic tumors International Collaboration (CHIC).

BACKGROUND: Liver tumors are rare in children with histologic heterogeneity that makes diagnosis challenging. Systematic histopathological review, performed as part of collaborative therapeutic protocols, identified relevant histologic subtypes that are important to distinguish. The Children's Hepatic tumors International Collaboration (CHIC) was established to study pediatric liver tumors on a global scale and led to establishment of a provisional consensus classification for use in international clinical trials. The current study is the validation of this initial classification and first large-scale application by international expert reviewers. PROCEDURE: The CHIC initiative includes data from 1605 children treated on eight multicenter hepatoblastoma (HB) trials. Review of 605 available tumors was performed by seven expert pathologists from three consortia (US, EU, Japan). Cases with discordant diagnoses were collectively reviewed to reach a final consensus diagnosis. RESULTS: Of 599 cases with sufficient material for review, 570 (95.2%) were classified as HB by all consortia, and 29 (4.8%) as non-HB, which included "hepatocellular neoplasm, NOS" and malignant rhabdoid tumors. 453 of 570 HBs were classified as epithelial by final consensus. Some patterns (i.e., small cell undifferentiated, macrotrabecular, cholangioblastic) were selectively identified by reviewers from different consortia. All consortia identified a similar number of mixed epithelial-mesenchymal HB. CONCLUSIONS: This study represents the first large-scale application and validation of the pediatric malignant hepatocellular tumors consensus classification. It is a valuable resource to train future generations of investigators on accurate diagnosis of these rare tumors and provides a framework for further international collaborative studies and refinement of the current classification of pediatric liver tumors.

Outcomes of Patients Treated for Hepatoblastoma with Low Alpha-Fetoprotein and/or Small Cell Undifferentiated Histology: A Report from the Children's Hepatic Tumors International Collaboration (CHIC).

Small cell undifferentiated (SCU) histology and alpha-fetoprotein (AFP) levels below 100 ng/mL have been reported as poor prognostic factors in hepatoblastoma (HB); subsequent studies reported SMARCB1 mutations in some SCU HBs confirming the diagnosis of rhabdoid tumor. The Children's Hepatic tumors International Collaboration (CHIC) database was queried for patients with HB who had AFP levels less than 100 ng/mL at diagnosis or were historically diagnosed as SCU HBs. Seventy-three of 1605 patients in the CHIC database were originally identified as SCU HB, HB with SCU component, or HB with low AFP levels. Upon retrospective review, they were re-classified as rhabdoid tumors (n = 11), HB with SCU component (n = 41), and HB with low AFP (n = 14). Seven were excluded for erroneously low AFP levels. Overall survival was 0% for patients with rhabdoid tumors, 76% for patients with HB with SCU component, and 64% for patients with HB with AFP less than 100 ng/mL. Patients with HB with SCU component or low AFP should be assessed for SMARCB1 mutations and, if confirmed, treated as rhabdoid tumors. When rhabdoid tumors are excluded, the presence of SCU component and low AFP at diagnosis were not associated with poor prognosis in patients diagnosed with HB.

Optic tract edema in craniopharyngioma as a predictor of BRAFV600E mutation presence.

OBJECTIVE: the advent of BRAF inhibitors for preoperative treatment of craniopharyngioma has necessitated the identification of BRAFV600E status. Hence, we investigated predictors of BRAFV600E mutation in craniopharyngiomas. METHODS: this retrospective study utilized data from 30 patients who were newly diagnosed with craniopharyngioma between 2011 and 2021. Magnetic resonance imaging (MRI) and computed tomography were performed within 1 week prior to surgery. Genetic analysis for BRAF mutation was performed using the Oncomine next-generation sequencing panel or Sanger sequencing. The relationship between BRAF mutation and demographic data, endocrinological function and tumour characteristics on imaging was assessed. RESULTS: tumour tissue carried the BRAFV600E mutation in nine patients. There was no significant difference in age, sex, or presence of hormonal dysfunction amongst patients with and without the BRAFV600E mutation in the tumour. Most tumours with the BRAFV600E mutation were histologically categorized as papillary craniopharyngioma (P = 0.0005), and were solid (P = 0.0002) and supra-diaphragmatic (P = 0.0033) on MRI. BRAFV600E tumours were more frequently associated with optic tract edema than wild-type tumour s (55.6 vs. 0%, P = 0.0009) and all tumour s with optic tract edema carried the BRAFV600E mutation. Optic tract edema was not associated with tumour volume, cysts, or preoperative pituitary function. CONCLUSIONS: in craniopharyngiomas, the presence of optic tract edema can predict the presence of BRAFV600E mutation with a positive predictive value of 100%. The finding should be verified in larger prospective cohorts and multivariate regression analysis.

Single-cell next-generation sequencing of circulating tumor cells in patients with neuroblastoma.

Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2+ CD90+ CD45- CD235a- DAPI- cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.

Usefulness of central radiologic review in clinical trials of children with hepatoblastoma.

BACKGROUND: No previous research papers have reported a comparative survey of local radiologic diagnoses and central review in children with hepatoblastoma. OBJECTIVE: To evaluate the utility of central review of children with hepatoblastoma enrolled in a clinical trial. MATERIALS AND METHODS: The study included 91 children enrolled in a clinical trial conducted by the Japanese Study Group for Pediatric Liver Tumor. We compared the results of the initial pre-treatment extent of tumor (PRETEXT) disease staging performed at local sites with the results obtained on central review to determine the concurrence rates for tumor staging and additional criteria. RESULTS: The concurrence rate for PRETEXT staging was 70%. As the stage increased, the concurrence rate decreased. Using additional criteria, central review identified 143 lesions (157.1%), about 1.8 times higher than the number identified for the local site diagnoses. The additional criterion found most often on central review was "multifocal lesion" (n=19). The concurrence rate for lung metastases was high. However, our central review found many false-positive assertions of hepatic vein lesions, portal vein invasion and extrahepatic lesions among the local site diagnoses. CONCLUSION: In a clinical trial of hepatoblastoma, central review provided a more precise diagnosis than local site diagnoses with respect to severe PRETEXT stages III and IV cases and other cases including hepatic and portal vein invasion. The central review process appears to be effective and essential for improving the quality of clinical trials.

Determination of the umbilicus position in Japanese children.

BACKGROUND: Although there have been many reports concerning the normal position of the umbilicus, the measurements were performed from the surface of the body in all cases. We examined computed tomography (CT) images to determine the accurate position of the umbilicus in children. METHODS: We retrospectively examined the CT data of 120 Japanese children (60 boys, 60 girls). The angle between both iliac crests to the umbilicus (IU angle), the angle between both anterior superior iliac spines and the umbilicus (AU angle), and the ratio of the length from the xiphoid process to the umbilicus and length from the umbilicus to the pubic symphysis were measured. RESULTS: The mean AU angle was 33.7° ± 5.1°, showing the least data variations. A significant difference was noted in the AU angle between boys and girls (32.7° ± 4.6° and 34.6° ± 5.4° respectively; p = 0.04). When we defined the position of the umbilicus as an AU angle of 33° in boys and 35° in girls, 115 children (95.8%) fell within ±10°. CONCLUSIONS: The AU angle is the preferable predictor of the umbilicus position in children.

A new surgical technique for short bowel syndrome.

OBJECTIVE: Short bowel syndrome (SBS) is a severe intestinal disease that causes malabsorption. Long-term parental nutrition therapy induces infection and liver failure. For the surgical management of intestinal rehabilitation, the intestinal loop lengthening method and serial transverse enteroplasty (STEP) method have been reported, although their effects have proven limited. We herein report a new surgical technique, Saeki-Spiral-Shark (3S) method for SBS using biomimetics of shark intestine. METHODS: In the 3S method, a spiral valve is formed inside the intestine by external sutures. Using a 25 cm length intestinal organ model, we performed both the 3S method and STEP procedure. We then compared the length and fluid passage times of the subsequently formed intestine. RESULTS: After the 3S method was performed, the length of the intestinal model changed to 22 cm, and after the STEP procedure, that was elongated to 30 cm. Although the water passage times did not change markedly, the semi-digestive nutritional supplement passage time slowed down in the model with the 3S method. There was slight leakage in the STEP procedure model. CONCLUSIONS: The 3S method is a unique method of treating SBS based on biomimetics. This procedure does not require an incision of the intestine, which thereby enabling clean and less-invasive surgery. We plan to conduct animal experiments in the future.

In Vitro Transfection of Up-Regulated Genes Identified in Favorable-Outcome Neuroblastoma into Cell Lines.

We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence.

Oncogenic Role of ADAM32 in Hepatoblastoma: A Potential Molecular Target for Therapy.

Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More effective and safer drugs are needed that are based on molecular mechanisms. A disintegrin and metalloproteases (ADAMs) are expressed with high frequency in various human carcinomas and play an important role in cancer progression. In this study, we analyzed expression of ADAMs in HBL with a cDNA microarray dataset and found that the expression level of ADAM32 is particularly high. To investigate the role of ADAM32 in cancer, forced expression or knockdown experiments were conducted with HepG2 and HBL primary cells. Colony formation, cell migration and invasion, and cell viability were increased in HepG2 expressing ADAM32, whereas knockdown of ADAM32 induced a decrease in these cellular functions. Quantitative RT-PCR demonstrated an association between ADAM32 expression and the expression of genes related to cancer stem cells and epithelial-mesenchymal transition (EMT), suggesting a role of ADAM32 in cancer stemness and EMT. Furthermore, knockdown of ADAM32 increased cisplatin-induced apoptosis, and this effect was attenuated by a caspase-8 inhibitor, suggesting that ADAM32 plays a role in extrinsic apoptosis signaling. We conclude that ADAM32 plays a crucial role in progression of HBL, so it might be a promising molecular target in anticancer therapy.