Vincristine/irinotecan/temsirolimus upfront window treatment of high-risk hepatoblastoma: A report from the Children's Oncology Group AHEP0731 Study Committee.

BACKGROUND: Survival for children with metastatic hepatoblastoma (HB) remains suboptimal. We report the response rate and outcome of two courses of vincristine/irinotecan/temsirolimus (VIT) in children with high-risk (HR)/metastatic HB. PROCEDURES: Patients with newly diagnosed HB received HR window chemotherapy if they had metastatic disease or a serum alpha-fetoprotein (AFP) level less than 100 ng/mL. Patients received vincristine (days 1 and 8), irinotecan (days 1-5), and temsirolimus (days 1 and 8). Cycles were repeated every 21 days. Responders had either a 30% decrease using RECIST (Response Evaluation Criteria in Solid Tumors) criteria OR a 90% (>1 log10 decline) AFP decline after two cycles. Responders received two additional cycles of VIT intermixed with six cycles of cisplatin/doxorubicin/5-fluorouracil/vincristine (C5VD). Nonresponders received six cycles of C5VD alone. RESULTS: Thirty-six eligible patients enrolled on study. The median age at enrollment was 27 months (range: 7-170). Seventeen of 36 patients were responders (RECIST and AFP = 3, RECIST only = 4, AFP only = 10). The median AFP at diagnosis was 222,648 ng/mL and the median AFP following two VIT cycles was 19,262 ng/mL. Three-year event-free survival was 47% (95% confidence interval [CI]: 30%-62%), while overall survival was 67% (95% CI: 49%-80%). CONCLUSION: VIT did not achieve the study efficacy endpoint. Temsirolimus does not improve the response rate seen in patients treated with vincristine and irinotecan (VI) alone as part of the initial treatment regimen explored in this study. Additionally, AFP response may be a more sensitive predictor of disease response than RECIST in HB.

Biliary Atresia Remnants Revisited: Myogenesis, Hepatic Duct-Like Structures, and Fate of Peribiliary Glands.

PURPOSE AND CONTEXT: Proximal levels of excised remnants from youngest infants may reveal early features of biliary atresia (BA). METHOD: A targeted IHC survey was applied to 34 most proximal 2 levels in 17 BA remnants excised at age 10-74 days including 7 = <30 days old and 6 control hepatic ducts (HD). KEY RESULTS: Severity of inflammation and extent of active fibroplasia do not distinguish proximal remnants in younger (n = 7) and older (n = 10) infants. In 27/34 levels of 14/17 remnants, reactive stroma is focally SM-MHC-2 (+), marking smooth muscle myosin, termed reactive myogenesis (RM), that is absent in controls. RM facilitates identification of 3 novel hepatic duct remnants (HDR): an HD-like collagen collar lined by degenerating cholangiocytes (n = 5); erosion defects in loose reactive stroma (n = 14); solitary foci of hyperplastic squamoid epithelium (n = 4). Peribiliary glands are either hyperplastic or atretic and typically lack RM. CONCLUSION: Minimally inflammed end-stage lesions in BA remnants occur at youngest ages favoring prenatal onset. Three novel HDR are defined. RM, a useful surrogate for HDR, is a prevalent inappropriate stromal reaction in proximal remnants of uncertain biological significance. RM is the source of mature smooth muscle in BA remnants.

Outcomes of children with well-differentiated fetal hepatoblastoma treated with surgery only: Report from Children's Oncology Group Trial, AHEP0731.

BACKGROUND: Hepatoblastoma (HB) requires surgical resection for cure, but only 20-30% of patients have resectable disease at diagnosis. Patients who undergo partial hepatectomy at diagnosis have historically received 4-6 cycles of adjuvant chemotherapy; however, those with 100% well-differentiated fetal histology (WDF) have been observed to have excellent outcomes when treated with surgery alone. PATIENTS AND METHODS: Patients on the Children's Oncology Group non randomized, multicenter phase III study, AHEP0731, were stratified based on Evan's stage, tumor histology, and serum alpha-fetoprotein level at diagnosis. Patients were eligible for the very low risk stratum of surgery and observation if they had a complete resection at diagnosis and rapid central histologic review demonstrated HB with 100% WDF histology. RESULTS: A total of 8 eligible patients were enrolled on study between September 14, 2009 and May 28, 2014. Outcome current to 06/30/2020 was used in this analysis. The median age at enrollment was 22.5 months (range: 8-84 months) and the median AFP at enrollment was 714 ng/ml (range: 18-77,747 ng/mL). With a median follow-up of 6.6 years (range: 3.6-9.8 years), the 5-year event-free (EFS) and overall survival (OS) were both 100%. CONCLUSION: This report supports that HB with 100% WDF histology completely resected at diagnosis is curable with surgery only. The development of evidence-based surgical guidelines utilizing criteria based on PRETEXT group, vascular involvement (annotation factors), tumor-specific histology and corresponding biology will be crucial for optimizing which patients are candidates for resection at diagnosis followed by observation. LEVEL OF EVIDENCE: Prognosis study, Level I evidence.

Hepatoblastomas with carcinoma features represent a biological spectrum of aggressive neoplasms in children and young adults.

BACKGROUND & AIMS: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS. METHODS: We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (>8). RESULTS: Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (∼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes. CONCLUSIONS: Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms. LAY SUMMARY: We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.

Intrahepatic Cholestasis, Refractory Epilepsy, Skeletal Dysplasia, Endocrine Failure, and Dysmorphic Features in a Child With a Monoallelic 2q24-32.2 Deletion Encompassing ABCB11.

We report a newborn who presented with multiple limb and facial anomalies, endocrine disorders, and progressively worsening low-GGT cholestasis. A liver biopsy revealed hepatocellular cholestasis with giant cell transformation. Immunohistochemical staining revealed complete absence of BSEP protein compared to control liver. A large 2q24-32.2 deletion leading to loss of 78 OMIM genes. Multiple structural anomalies, epilepsy and endocrine anomalies have been described with hemizygous loss of these genes. This deletion also resulted in complete heterozygous deletion of ABCB11, which encodes the bile salt export pump (BSEP). Genetic analysis did not reveal any pathogenic variants, deletions, or duplications in the other ABCB11 allele. A heterozygous variant in NR1H4, which causes the autosomal recessive progressive familial intrahepatic cholestasis type 5, was also detected. The possible explanations for the PFIC type 2 phenotype in heterozygous loss of ABCB11 include genetic modifiers or di-genic disease with a compound ABCB11 deletion and an NR1H4 missense variant; or undetected pathogenic variants in the other ABCB11 or NR1H4 alleles.

Doxorubicin in combination with cisplatin, 5-flourouracil, and vincristine is feasible and effective in unresectable hepatoblastoma: A Children's Oncology Group study.

BACKGROUND: The Children's Oncology Group (COG) adopted cisplatin, 5-flourouracil, and vincristine (C5V) as standard therapy after the INT-0098 legacy study showed statistically equivalent survival but less toxicity in comparison with cisplatin and doxorubicin. Subsequent experience demonstrated doxorubicin to be effective in patients with recurrent disease after C5V, and this suggested that it could be incorporated to intensify therapy for patients with advanced disease. METHODS: In this nonrandomized, phase 3 COG trial, the primary aim was to explore the feasibility and toxicity of a novel therapeutic cisplatin, 5-flourouracil, vincristine, and doxorubicin (C5VD) regimen with the addition of doxorubicin to C5V for patients considered to be at intermediate risk. Patients were eligible if they had unresectable, nonmetastatic disease. Patients with a complete resection at diagnosis and local pathologic evidence of small cell undifferentiated histology were also eligible for an assessment of feasibility. RESULTS: One hundred two evaluable patients enrolled between September 14, 2009, and March 12, 2012. Delivery of C5VD was feasible and tolerable: the mean percentages of the target doses delivered were 96% (95% CI, 94%-97%) for cisplatin, 96% (95% CI, 94%-97%) for 5-fluorouracil, 95% (95% CI, 93%-97%) for doxorubicin, and 90% (95% CI, 87%-93%) for vincristine. Toxicity was within expectations, with death as a first event in 1 patient. The most common adverse events were febrile neutropenia (n = 55 [54%]), infection (n = 48 [47%]), mucositis (n = 31 [30%]), hypokalemia (n = 39 [38%]), and elevated aspartate aminotransferase (n = 28 [27%]). The 5-year event-free and overall survival rates for the 93 patients who did not have complete resection at diagnosis were 88% (95% CI, 79%-93%) and 95% (95% CI, 87%-98%), respectively. CONCLUSIONS: The addition of doxorubicin to the previous standard regimen of C5V is feasible, tolerable, and efficacious, and this suggests that C5VD is a good regimen for future clinical trials.

Small Cell Undifferentiated Histology Does Not Adversely Affect Outcome in Hepatoblastoma: A Report From the Children's Oncology Group (COG) AHEP0731 Study Committee.

PURPOSE: Small cell undifferentiated (SCU) histology in hepatoblastoma (HB) tumors has historically been associated with a poor prognosis. Tumors from patients enrolled on Children's Oncology Group (COG) study AHEP0731 underwent institutional and central pathologic review for identification of SCU histology. PATIENTS AND METHODS: Patients with SCU histology identified at the local treating institution who had otherwise low-risk tumors were upstaged to the intermediate-risk treatment stratum, whereas those only identified by retrospective central review were treated per the local institution as low-risk. Patients with otherwise intermediate- or high-risk tumors remained in that treatment stratum, respectively. Central review was to be performed for all tissue samples obtained at any time point. Treatment was per local review, whereas analysis of outcome was based on central review. RESULTS: Thirty-five patients had some elements (1%-25%) of SCU identified on central review of diagnostic specimens. All but two patient tissue sample retained nuclear INI1 expression. The presence of SCU histology did not correlate with age, alpha-fetoprotein level at diagnosis, or sex. The presence of SCU did not affect event-free survival (EFS). EFS at 5 years for patients with low-risk, intermediate-risk, and high-risk with SCU HB was 86% (95% CI, 33 to 98), 81% (95% CI, 57 to 92), and 29% (95% CI, 4 to 61), respectively, compared with EFS at 5 years for patients without SCU enrolled with low-risk, intermediate-risk, and high-risk of 87% (95% CI, 72 to 95), 88% (95% CI, 79 to 94), and 55% (95% CI, 32 to 74; P = .17), respectively. CONCLUSION: The presence of SCU histology in HB does not appear to adversely affect outcome. Future studies should be able to treat patients with SCU HB according to risk stratification without regard to the presence of SCU histology.

Presentation and Outcomes of Infants With Idiopathic Cholestasis: A Multicenter Prospective Study.

OBJECTIVES: The aim of the study was to determine the frequency and natural history of infantile idiopathic cholestasis (IC) in a large, prospective, multicenter cohort of infants. METHODS: We studied 94 cholestatic infants enrolled up to 6 months of age in the NIDDK ChiLDReN (Childhood Liver Disease Research Network) "PROBE" protocol with a final diagnosis of IC; they were followed up to 30 months of age. RESULTS: Male sex (66/94; 70%), preterm birth (22/90 with data; 24% born at < 37 weeks' gestational age), and low birth weight (25/89; 28% born at <2500 g) were frequent, with no significant differences between outcomes. Clinical outcomes included death (n = 1), liver transplant (n = 1), biochemical resolution (total bilirubin [TB] ≤1 mg/dL and ALT < 35 U/L; n = 51), partial resolution (TB > 1 mg/dL and/or ALT > 35 U/L; n = 7), and exited healthy (resolved disease per study site report but without documented biochemical resolution; n = 34). Biochemical resolution occurred at median of 9 months of age. GGT was <100 U/L at baseline in 34 of 83 participants (41%). CONCLUSIONS: Frequency of IC and of death or liver transplant was less common in this cohort than in previously published cohorts, likely because of recent discovery and diagnosis of genetic etiologies of severe/persistent cholestasis that previously were labeled as idiopathic. Preterm birth and other factors associated with increased vulnerability in neonates are relatively frequent and may contribute to IC. Overall outcome in IC is excellent. Low/normal GGT was common, possibly indicating a role for variants in genes associated with low-GGT cholestasis-this warrants further study.

TFEB regulates murine liver cell fate during development and regeneration.

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer.

NR1H4-related Progressive Familial Intrahepatic Cholestasis 5: Further Evidence for Rapidly Progressive Liver Failure.

Pathogenic sequence variants in the nuclear bile acid receptor FXR, encoded by NR1H4, have been reported in a small number of children with low-γ-glutamyl transferase (GGT) cholestasis progressing to liver failure. We describe 3 additional children from 2 unrelated families with cholestasis and liver failure because of pathologic variants in NR1H4. One patient underwent liver transplantation and has had good clinical outcomes in 6 years of follow-up. Although that patient has biochemical evidence of increased bile acid synthetic activity, he has not experienced post-transplant diarrhea or allograft steatosis, as has been reported among other transplanted patients.

Diagnostic Yield of Newborn Screening for Biliary Atresia Using Direct or Conjugated Bilirubin Measurements.

Importance: Treating biliary atresia in newborns earlier can delay or prevent the need for liver transplant; however, treatment typically occurs later because biliary atresia is difficult to detect during its early stages. Objective: To determine the diagnostic yield of newborn screening for biliary atresia with direct or conjugated bilirubin measurements and to evaluate the association of screening implementation with clinical outcomes. Design, Setting, and Participants: A cross-sectional screening study of 124 385 infants born at 14 Texas hospitals between January 2015 and June 2018; and a pre-post study of 43 infants who underwent the Kasai portoenterostomy as treatment for biliary atresia at the region's largest pediatric hepatology center before (January 2008-June 2011) or after (January 2015-June 2018) screening implementation. Final follow-up occurred on July 15, 2019. Exposures: Two-stage screening with direct or conjugated bilirubin measurements. In stage 1, all newborns were tested within the first 60 hours of life, with a positive screening result defined as bilirubin levels exceeding derived 95th percentile reference intervals. In stage 2, infants who had a positive screening result in stage 1 were retested at or before the 2-week well-child visit, with a positive screening result defined as bilirubin levels greater than the stage 1 result or greater than 1 mg/dL. Main Outcomes and Measures: The primary outcomes of the screening study were sensitivity, specificity, positive predictive value, and negative predictive value based on infants testing positive in both stages. The reference standard was biliary atresia diagnosed at the region's pediatric hepatology centers. The primary outcome of the pre-post study was the age infants underwent the Kasai portoenterostomy for treatment of biliary atresia. Results: Of 124 385 newborns in the screening study, 49.2% were female, 87.6% were of term gestational age, 70.0% were white, and 48.1% were Hispanic. Screening identified the 7 known infants with biliary atresia with a sensitivity of 100% (95% CI, 56.1%-100.0%), a specificity of 99.9% (95% CI, 99.9%-99.9%), a positive predictive value of 5.9% (95% CI, 2.6%-12.2%), and a negative predictive value of 100.0% (95% CI, 100.0%-100.0%). In the pre-post study, 24 infants were treated before screening implementation and 19 infants were treated after screening implementation (including 6 of 7 from the screening study, 7 from screening at nonstudy hospitals, and 6 from referrals because of clinical symptoms). The age infants underwent the Kasai portoenterostomy was significantly younger after screening was implemented (mean age, 56 days [SD, 19 days] before screening implementation vs 36 days [SD, 22 days] after screening implementation; between-group difference, 19 days [95% CI, 7-32 days]; P = .004). Conclusions and Relevance: Newborn screening with direct or conjugated bilirubin measurements detected all known infants with biliary atresia in the study population, although the 95% CI around the sensitivity estimate was wide and the study design did not ensure complete ascertainment of false-negative results. Research is needed in larger populations to obtain more precise estimates of diagnostic yield and to better understand the clinical outcomes and cost-effectiveness of this screening approach.

Nucleostemin Modulates Outcomes of Hepatocellular Carcinoma via a Tumor Adaptive Mechanism to Genomic Stress.

Hepatocellular carcinomas (HCC) are adapted to survive extreme genomic stress conditions imposed by hyperactive DNA replication and genotoxic drug treatment. The underlying mechanisms remain unclear, but may involve intensified DNA damage response/repair programs. Here, we investigate a new role of nucleostemin (NS) in allowing HCC to survive its own malignancy, as NS was previously shown to promote liver regeneration via a damage repair mechanism. We first established that a higher NS transcript level correlates with high-HCC grades and poor prognostic signatures, and is an independent predictor of shorter overall and progression-free survival specifically for HCC and kidney cancer but not for others. Immunostaining confirmed that NS is most abundantly expressed in high-grade and metastatic HCCs. Genome-wide analyses revealed that NS is coenriched with MYC target and homologous recombination (HR) repair genes in human HCC samples and functionally intersects with those involved in replication stress response and HR repair in yeasts. In support, NS-high HCCs are more reliant on the replicative/oxidative stress response pathways, whereas NS-low HCCs depend more on the mTOR pathway. Perturbation studies showed NS function in protecting human HCC cells from replication- and drug-induced DNA damage. Notably, NS depletion in HCC cells increases the amounts of physical DNA damage and cytosolic double-stranded DNA, leading to a reactive increase of cytokines and PD-L1. This study shows that NS provides an essential mechanism for HCC to adapt to high genomic stress for oncogenic maintenance and propagation. NS deficiency sensitizes HCC cells to chemotherapy but also triggers tumor immune responses. IMPLICATIONS: HCC employs a novel, nucleostemin (NS)-mediated-mediated adaptive mechanism to survive high genomic stress conditions, a deficiency of which sensitizes HCC cells to chemotherapy but also triggers tumor immune responses.

Hepatic Tumor Formation in Adult Mice Developmentally Exposed to Organotin.

BACKGROUND: Tributyltin (TBT) is a persistent and bioaccumulative environmental toxicant. Developmental exposure to TBT has been shown to cause fatty liver disease (steatosis), as well as increased adiposity in many species, leading to its characterization as an obesogen. OBJECTIVE: We aimed to determine the long-term effects of developmental TBT exposure on the liver. METHODS: C57BL/6J mice were exposed to a dose of TBT (0.5mg/kg body weight per day; 3.07µM) below the current developmental no observed adverse effect level (NOAEL) via drinking water, or drinking water alone, provided to the dam from preconception through lactation. Sires were exposed during breeding and lactation. Pups from two parity cycles were included in this study. Animals were followed longitudinally, and livers of offspring were analyzed by pathological evaluation, immunohistochemistry, immunoblotting, and RNA sequencing. RESULTS: Developmental exposure to TBT led to increased adiposity and hepatic steatosis at 14 and 20 weeks of age and increased liver adenomas at 45 weeks of age in male offspring. Female offspring displayed increased adiposity as compared with males, but TBT did not lead to an increase in fatty liver or tumor development in female offspring. Liver tumors in male mice were enriched in pathways and gene signatures associated with human and rodent nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). This includes down-regulation of growth hormone receptor (GHR) and of STAT5 signaling, which occurred in response to TBT exposure and preceded liver tumor development. CONCLUSIONS: These data reveal a previously unappreciated ability of TBT to increase risk for liver tumorigenesis in mice in a sex-specific manner. Taken together, these findings provide new insights into how early life environmental exposures contribute to liver disease in adulthood. https://doi.org/10.1289/EHP5414.

Evaluation of the diagnostic biopsy approach for children with hepatoblastoma: A report from the children's oncology group AHEP 0731 liver tumor committee.

BACKGROUND: The histopathological assessment of pediatric liver tumors at presentation is critical to establish a diagnosis, guide treatment, and collect appropriate research samples. The purpose of this study was to evaluate complications associated with different approaches to liver biopsy for newly diagnosed hepatoblastoma. METHODS: Children with hepatoblastoma were enrolled on Children's Oncology Group study AHEP0731 (September 2009-March 2012). This analysis evaluated the study cohort of initially unresectable patients who therefore underwent a biopsy procedure at diagnosis. The primary endpoint was clinically significant postbiopsy hemorrhage, defined as requiring red blood cell transfusion. RESULTS: We identified 121 children who underwent open (n = 76, 63%), laparoscopic (n = 17, 14%), or percutaneous (n = 28, 23%) liver biopsies. All biopsy procedures yielded adequate tissue for diagnosis. Postbiopsy hemorrhage requiring transfusion occurred after 26% (n = 31) of biopsies. Need for blood product transfusion most frequently occurred following open (n = 27/76, 36%) and laparoscopic (n = 4/17, 24%) biopsies, compared with percutaneous (n = 0/28, 0%) biopsies (p < 0.01). CONCLUSIONS: Pretreatment biopsy of pediatric liver tumors via a percutaneous approach yielded the lowest frequency of clinically significant hemorrhage requiring transfusion, without evidence of sacrificing diagnostic accuracy. LEVEL OF EVIDENCE: Level I.

Sox9 Is a Modifier of the Liver Disease Severity in a Mouse Model of Alagille Syndrome.

BACKGROUND AND AIMS: Alagille syndrome (ALGS) is a multisystem developmental disorder characterized by bile duct (BD) paucity, caused primarily by haploinsufficiency of the Notch ligand jagged1. The course of the liver disease is highly variable in ALGS. However, the genetic basis for ALGS phenotypic variability is unknown. Previous studies have reported decreased expression of the transcription factor SOX9 (sex determining region Y-box 9) in late embryonic and neonatal livers of Jag1-deficient mice. Here, we investigated the effects of altering the Sox9 gene dosage on the severity of liver disease in an ALGS mouse model. APPROACH AND RESULTS: Conditional removal of one copy of Sox9 in Jag1+/- livers impairs the biliary commitment of cholangiocytes and enhances the inflammatory reaction and liver fibrosis. Loss of both copies of Sox9 in Jag1+/- livers further worsens the phenotypes and results in partial lethality. Ink injection experiments reveal impaired biliary tree formation in the periphery of P30 Jag1+/- livers, which is improved by 5 months of age. Sox9 heterozygosity worsens the P30 biliary tree phenotype and impairs the partial recovery in 5-month-old animals. Notably, Sox9 overexpression improves BD paucity and liver phenotypes in Jag1+/- mice without ectopic hepatocyte-to-cholangiocyte transdifferentiation or long-term liver abnormalities. Notch2 expression in the liver is increased following Sox9 overexpression, and SOX9 binds the Notch2 regulatory region in the liver. Histological analysis shows a correlation between the level and pattern of SOX9 expression in the liver and outcome of the liver disease in patients with ALGS. CONCLUSIONS: Our results establish Sox9 as a dosage-sensitive modifier of Jag1+/- liver phenotypes with a permissive role in biliary development. Our data further suggest that liver-specific increase in SOX9 levels is a potential therapeutic approach for BD paucity in ALGS.

Minimal adjuvant chemotherapy for children with hepatoblastoma resected at diagnosis (AHEP0731): a Children's Oncology Group, multicentre, phase 3 trial.

BACKGROUND: Hepatoblastoma treatment with curative intent requires surgical resection, but only about a third of newly diagnosed patients with hepatoblastoma have resectable disease at diagnosis. Patients who have upfront resection typically receive a total of 4-6 cycles of adjuvant chemotherapy post-surgery, with the combination of cisplatin, fluorouracil, and vincristine. We aimed to investigate whether event-free survival in children with hepatoblastoma who had complete resection at diagnosis could be maintained with two cycles of adjuvant chemotherapy. METHODS: In this Children's Oncology Group, multicentre, phase 3 trial, patients were enrolled in four risk groups on the basis of Evans surgical stage, tumour histology, and levels of α-fetoprotein at diagnosis to receive risk-adapted therapy. Here, we report on the low-risk stratum of the trial. Eligible patients were younger than 21 years and had histologically confirmed, stage I or II hepatoblastoma without 100% pure fetal stage I or small-cell undifferentiated histology; elevated serum α-fetoprotein level (>100 ng/mL); a complete resection at diagnosis; at least 50% Karnofsky (patients >16 years) or Lansky (patients ≤16 years) performance status; and had received no previous chemotherapy or other hepatoblastoma-directed therapy. Patients received two 21-day cycles of cisplatin, fluorouracil, and vincristine within 42 days of resection, consisting of cisplatin (100 mg/m2 per dose or 3·3 mg/kg per dose for children <10 kg) intravenously over 6 h on day 1; fluorouracil (600 mg/m2 per dose or 20 mg/kg per dose for children <10 kg) intravenous push on day 2; and vincristine (1·5 mg/m2 per day to a maximum dose of 2 mg, or 0·05 mg/kg per day for children <10 kg) intravenous push on days 2, 9, and 16. The primary outcome was investigator-assessed event-free survival. As prespecified by protocol, we analysed the primary endpoint 6 years after enrolment (cutoff date June 30, 2017). This trial is registered with ClinicalTrials.gov, number NCT00980460, and is now permanently closed to accrual. FINDINGS: Between May 18, 2010, and May 28, 2014, 51 patients in 32 centres in two countries were enrolled into the low-risk stratum of this trial, of whom 49 received c hemotherapy treatment after surgery and were evaluable for activity and safety. Median follow-up time for all evaluable patients was 42 months (IQR 36-62). 4-year event-free survival was 92% (95% CI 79-97) and 5-year event-free survival was 88% (72-95). Two (4%) of 49 patients had surgical complications (bile leaks). The most common grade 3-4 adverse events were febrile neutropenia in seven (14%) patients, decreased neutrophil count in three (6%) patients, infections in four (8%) patients, and diarrhoea in four (8%) patients. Ototoxicity occurred in one (2%) patient. One (2%) patient of the three who relapsed in this cohort died from disease. Two (4%) patients died in clinical remission after therapy discontinuation. One patient died of pneumonia and bacterial sepsis 1 year after therapy discontinuation and another patient died of unrelated causes 57 months after therapy completion. There were no treatment-related deaths. INTERPRETATION: Minimal postoperative chemotherapy with two cycles of cisplatin, fluorouracil, and vincristine can ensure disease control in patients with hepatoblastoma resected at diagnosis. Our results show that dose reduction of ototoxic agents is a safe, effective treatment for these children. FUNDING: National Institutes of Health.

Characterization of pediatric hepatocellular carcinoma reveals genomic heterogeneity and diverse signaling pathway activation.

BACKGROUND: Pediatric hepatocellular carcinoma (HCC) is a rare liver tumor in children with a poor prognosis. Comprehensive molecular profiling to understand the underlying genomic drivers of this tumor has not been completed, and it is unclear whether nonfibrolamellar pediatric HCC is more genomically similar to hepatoblastoma or adult HCC. PROCEDURE: To characterize the molecular landscape of these tumors, we analyzed a cohort of 15 pediatric non-FL-HCCs by sequencing a panel of cancer-associated genes and conducting copy-number and gene-expression analyses. RESULTS: We detected multiple types of molecular alterations in Wnt signaling genes, including APC inversion, AMER1 somatic mutation, and most commonly CTNNB1 intragenic deletions. There were multiple alterations to the telomerase pathway via TERT activation or ATRX mutation. Therapeutically targetable activating mutations in MAPK/ERK signaling pathway genes, including MAPK1 and BRAF, were detected in 20% of tumors. TP53 mutations occurred far less frequently in our pediatric HCC cohort than reported in adult cohorts. Tumors arising in children with underlying liver disease were found to be molecularly distinct from the remainder and lacking detectable oncogenic drivers, as compared with those arising in patients without a history of underlying liver disease; the majority of both types were positive for glypican-3, another potential therapeutic target. CONCLUSION: Our study revealed pediatric HCC to be a molecularly heterogeneous group of tumors. Those non-FL-HCC tumors arising in the absence of underlying liver disease harbor genetic alterations affecting multiple cancer pathways, most notably Wnt signaling, and share some characteristics with adult HCC.

Growth Factor-Independent 1 Is a Tumor Suppressor Gene in Colorectal Cancer.

Colorectal cancer is the third most common cancer and the third leading cause of cancer death in the United States. Growth factor-independent 1 (GFI1) is a zinc finger transcriptional repressor responsible for controlling secretory cell differentiation in the small intestine and colon. GFI1 plays a significant role in the development of human malignancies, including leukemia, lung cancer, and prostate cancer. However, the role of GFI1 in colorectal cancer progression is largely unknown. Our results demonstrate that RNA and protein expression of GFI1 are reduced in advanced-stage nonmucinous colorectal cancer. Subcutaneous tumor xenograft models demonstrated that the reexpression of GFI1 in 4 different human colorectal cancer cell lines inhibits tumor growth. To further investigate the role of Gfi1 in de novo colorectal tumorigenesis, we developed transgenic mice harboring a deletion of Gfi1 in the colon driven by CDX2-cre (Gfi1F/F; CDX2-cre) and crossed them with ApcMin/+ mice (ApcMin/+; Gfi1F/F; CDX2-cre). Loss of Gfi1 significantly increased the total number of colorectal adenomas compared with littermate controls with an APC mutation alone. Furthermore, we found that compound (ApcMin/+; Gfi1F/F; CDX2-cre) mice develop larger adenomas, invasive carcinoma, as well as hyperplastic lesions expressing the neuroendocrine marker chromogranin A, a feature that has not been previously described in APC-mutant tumors in mice. Collectively, these results demonstrate that GFI1 acts as a tumor suppressor gene in colorectal cancer, where deficiency of Gfi1 promotes malignancy in the colon. IMPLICATIONS: These findings reveal that GFI1 functions as a tumor suppressor gene in colorectal tumorigenesis.

Identification of Polycystic Kidney Disease 1 Like 1 Gene Variants in Children With Biliary Atresia Splenic Malformation Syndrome.

Biliary atresia (BA) is the most common cause of end-stage liver disease in children and the primary indication for pediatric liver transplantation, yet underlying etiologies remain unknown. Approximately 10% of infants affected by BA exhibit various laterality defects (heterotaxy) including splenic abnormalities and complex cardiac malformations-a distinctive subgroup commonly referred to as the biliary atresia splenic malformation (BASM) syndrome. We hypothesized that genetic factors linking laterality features with the etiopathogenesis of BA in BASM patients could be identified through whole-exome sequencing (WES) of an affected cohort. DNA specimens from 67 BASM subjects, including 58 patient-parent trios, from the National Institute of Diabetes and Digestive and Kidney Diseases-supported Childhood Liver Disease Research Network (ChiLDReN) underwent WES. Candidate gene variants derived from a prespecified set of 2,016 genes associated with ciliary dysgenesis and/or dysfunction or cholestasis were prioritized according to pathogenicity, population frequency, and mode of inheritance. Five BASM subjects harbored rare and potentially deleterious biallelic variants in polycystic kidney disease 1 like 1 (PKD1L1), a gene associated with ciliary calcium signaling and embryonic laterality determination in fish, mice, and humans. Heterozygous PKD1L1 variants were found in 3 additional subjects. Immunohistochemical analysis of liver from the one BASM subject available revealed decreased PKD1L1 expression in bile duct epithelium when compared to normal livers and livers affected by other noncholestatic diseases. Conclusion: WES identified biallelic and heterozygous PKD1L1 variants of interest in 8 BASM subjects from the ChiLDReN data set; the dual roles for PKD1L1 in laterality determination and ciliary function suggest that PKD1L1 is a biologically plausible, cholangiocyte-expressed candidate gene for the BASM syndrome.