Human liver organoids for disease modeling of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

Survival and Scoliosis Following Resection of Chest Wall Tumors in Children and Adolescents: A Single-center Retrospective Analysis.

OBJECTIVE: We reviewed our experience with pediatric chest wall tumors (CWTs) to identify variables associated with survival, scoliosis development, and need for corrective scoliosis surgery. BACKGROUND: Chest wall neoplasms in children or adolescents are rare. Consequently, there are few large series that detail survival or quality of life indicators, like scoliosis. METHODS: Medical records were reviewed for all chest wall resections for primary and metastatic CWT performed from October 1, 1986 to September 30, 2016 on patients 21 years or younger at diagnosis. Kaplan-Meier distributions were compared using the log-rank test. Variables correlated with survival, scoliosis development, or need for corrective surgeries were analyzed using competing-risk analysis. RESULTS: Seventy-six cases [57 (75%) primary, 19 (25%) metastatic] were identified. Median age at diagnosis was 15.6 years (range: 0.5-21 years). Tumor types were Ewing sarcoma family tumors (54%), other soft tissue sarcomas (21%), osteosarcoma (11%), rhabdomyosarcoma (7%), and other (8%). A median of 3 (range: 1-5) contiguous ribs were resected. Surgical reconstruction included composite Marlex mesh and methyl-methacrylate, Gore-Tex, or primary closure in 57%, 28%, and 14% of procedures, respectively. Overall 5-year survival was 61% (95% confidence interval: 50%-75%). Scoliosis developed in 19 (25%) patients; 6 patients required corrective surgery. Variables associated with overall survival were the presence of metastatic disease at diagnosis, and whether the chest tumor itself was a primary or metastatic lesion. Younger age at chest wall resection was associated with the need for corrective surgery in patients who developed scoliosis. CONCLUSIONS: Among pediatric and adolescent patients with CWTs, survival depends primarily on the presence of metastases. Age, type of chest wall reconstruction, and tumor size are not associated with scoliosis development. Among patients who develop scoliosis, younger patients are more likely to require corrective surgery.

A Human Organoid Model of Aggressive Hepatoblastoma for Disease Modeling and Drug Testing.

Hepatoblastoma is the most common childhood liver cancer. Although survival has improved significantly over the past few decades, there remains a group of children with aggressive disease who do not respond to current treatment regimens. There is a critical need for novel models to study aggressive hepatoblastoma as research to find new treatments is hampered by the small number of laboratory models of the disease. Organoids have emerged as robust models for many diseases, including cancer. We have generated and characterized a novel organoid model of aggressive hepatoblastoma directly from freshly resected patient tumors as a proof of concept for this approach. Hepatoblastoma tumor organoids recapitulate the key elements of patient tumors, including tumor architecture, mutational profile, gene expression patterns, and features of Wnt/ß-catenin signaling that are hallmarks of hepatoblastoma pathophysiology. Tumor organoids were successfully used alongside non-tumor liver organoids from the same patient to perform a drug screen using twelve candidate compounds. One drug, JQ1, demonstrated increased destruction of liver organoids from hepatoblastoma tumor tissue relative to organoids from the adjacent non-tumor liver. Our findings suggest that hepatoblastoma organoids could be used for a variety of applications and have the potential to improve treatment options for the subset of hepatoblastoma patients who do not respond to existing treatments.

A novel image-based system for risk stratification in patients with desmoplastic small round cell tumor.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is an aggressive soft tissue sarcoma affecting children and young adults with 5-year overall survival (OS) of approximately 20%. Despite generally poor prognosis, long-term survival does occur. However, no evidence-based system exists to risk-stratify patients at diagnosis. METHODS: We retrospectively reviewed all DSRCT cases diagnosed at our institution between January 2000 and September 2016. Demographics, diagnostic imaging, and clinical data were reviewed. Univariate and multivariate Cox proportional hazard modeling was used to evaluate associations between imaging characteristics and OS. RESULTS: There were 130 patients (85% male; median age at presentation: 21.2 years) with confirmed DSRCT and sufficient imaging and clinical information for analysis. Median 5-year OS was 28% (95% CI: 19%-37%). In univariate analysis, shorter OS was associated with presence of liver lesions (hazard ratio [HR] 2.1, 95% CI: 1.28-3.45), chest lesions (HR 1.86, 95% CI: 1.11-3.1), and ascites (HR 1.69, 95% CI: 1.06-2.7). In multivariate analysis, liver involvement and ascites were predictive and were used to stratify risk (intermediate=no liver involvement or ascites; high=either liver involvement or ascites; very high=both liver involvement and ascites). Intermediate-risk patients had a 5-year survival of 61% (95% CI: 40%-76%) versus 16% (95% CI: 6%-29%) among high-risk patients and 8% (95% CI: 1%-29%) among very high risk patients. CONCLUSION: Patients with DSRCT can be risk-stratified at diagnosis based on specific imaging characteristics. TYPE OF STUDY: Retrospective study with no comparison group. LEVEL OF EVIDENCE: Level IV.

Non coding RNA analysis in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary liver cancer found in adolescents and young adults without underlying liver disease. A deletion of ~400 kD has been found in one copy of chromosome 19 in the tumor tissue of all patients tested. This produces a fusion of the genes DNAJB1 and PRKACA which, in turn, produces a chimeric transcript and protein. Transcriptomic analysis of the tumor has shown upregulation of various oncologically relevant pathways, including EGF/ErbB, Aurora Kinase A, pak21 and wnt. To explore other factors that may contribute to oncogenesis, we examined the microRNA (miRNA) and long non-coding RNA (lncRNA) expression in FLC. The non-coding RNA expression profile in tumor tissue samples is distinctly different from the adjacent normal liver and from other liver tumors. Furthermore, miRZip knock down or over expression of certain miRNAs led to changes in the levels of coding genes that recapitulated changes observed in FLC, suggesting mechanistically that the changes in the cellular levels of miRNA are not merely correlative. Thus, in addition to serving as diagnostic tools for FLC, non-coding RNAs may serve as therapeutic targets.

Treatment and outcome of adult-onset neuroblastoma.

Adult-onset neuroblastoma is rare and little is known about its biology and clinical course. There is no established therapy for adult-onset neuroblastoma. Anti-GD2 immunotherapy is now standard therapy in children with high-risk neuroblastoma; however, its use has not been reported in adults. Forty-four adults (18-71 years old) diagnosed with neuroblastoma between 1979 and 2015 were treated at Memorial Sloan Kettering Cancer Center. Five, 1, 5 and 33 patients had INSS stage 1, 2, 3 and 4 diseases, respectively. Genetic abnormalities included somatic ATRX (58%) and ALK mutations (42%) but not MYCN-amplification. In the 11 patients with locoregional disease, 10-year progression-free (PFS) and overall survival (OS) was 35.4 ± 16.1% and 61.4 ± 15.3%, respectively. Among 33 adults with stage 4 neuroblastoma, 7 (21%) achieved complete response (CR) after induction chemotherapy and/or surgery. Seven patients with primary refractory neuroblastoma (all with osteomedullary but no soft tissue disease) received anti-GD2 antibodies, mouse or humanized 3F8. Antibody-related adverse events were similar to those in children, response rate being 71.4%. In patients with stage 4 disease at diagnosis, 5-year PFS was 9.7± 5.3% and most patients who were alive with disease at 5 years died of neuroblastoma over the next 5 years, 10-year OS being only 19.0 ± 8.2%. Patients who achieved CR after induction had superior PFS and OS (p = 0.006, p = 0.031, respectively). Adult-onset neuroblastoma appeared to have different biology from pediatric or adolescent NB, and poorer outcome. Complete disease control appeared to improve long-term survival. Anti-GD2 immunotherapy was well tolerated and might be beneficial.

Urothelial neoplasms in pediatric and young adult patients: A large single-center series.

PURPOSE: Bladder cancer is the sixth most common cancer in the United States, but is exceedingly rare in young patients, leading to a lack of accepted standards for diagnosis, treatment, and surveillance. We review our institutional experience with bladder urothelial neoplasms in pediatric and young adult patients summarizing presentation, treatment, and outcomes. METHODS: Surgical pathology records at our institution were searched for cases of urothelial neoplasms among patients ≤25 years of age treated between January 1997 and September 2016. Cases submitted exclusively for pathology review were excluded. Diagnoses were confirmed based on pathologic examination using the 2004 World Health Organization classification system. RESULTS: Thirty-four patients were identified with a mean age of 21.1 years (range 8-25 years), and median follow-up was 25.1 months (1-187 months). The male to female ratio was 1.83:1. The most common presenting symptom was hematuria (n=26; 76%). Diagnoses were invasive urothelial carcinoma (n=3), noninvasive urothelial carcinoma (n=24), PUNLMP (n=6), and urothelial papilloma (n=1). Noninvasive lesions were resected by cystoscopy, after which 12% (n=4) experienced complications (grade II or greater). One patient with stage IV invasive disease at diagnosis died, and 2 patients developed recurrences. Of those with noninvasive carcinoma, 29% (n=7) required repeat cystoscopy soon after initial TURBT at outside institutions, and 17% (n=4) had tumors downgraded from high-grade to low-grade after pathology review. CONCLUSION: Hematuria is the most common sign of bladder neoplasia in children and young adults and should be investigated by cystoscopy. The majority of urothelial neoplasms in these patients are noninvasive and can be successfully treated with transurethral resection. LEVEL OF EVIDENCE: Level IV (Retrospective study with no comparison group).

Intracranial metastasis in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver malignancy in adolescents and young adults. Surgery is the mainstay of therapy for primary and metastatic disease. Most patients relapse, with development of both local and distant metastases. Brain metastases from solid tumors are rare in the pediatric and young adult population. Here, we document three patients with brain metastases from FLHCC, confirmed by histology and molecular characterization of the chimeric fusion DNAJB1-PRKACA, each necessitating neurosurgical intervention. These observations highlight the ability of FLHCC to metastasize to the brain and suggest the need for surveillance neuroimaging for patients with advanced-stage disease.

Thyroid neoplasms: incidental findings on extent of disease evaluation CT for other pediatric malignancies.

PURPOSE: We performed a retrospective analysis to evaluate the risk of thyroid cancer in incidental thyroid nodules (ITNs) discovered on CT in patients with a history of pediatric cancer. METHODS: With IRB approval we reviewed the records of pediatric oncology patients age ≤21y with newly detected thyroid nodules on surveillance CT of the neck, chest, chest/abdomen/pelvis, or PET/CT performed between April 2008 and March 2015. Patients with <6months of follow-up after incidental findings, a history of primary thyroid malignancy, or incomplete records were excluded. RESULTS: The final cohort (N=68) included 35 females and 33 males (mean age 16.0±4.3[SD] years) with a mean follow-up time of 3.7±1.9[SD] years after CT detection of ITN(s). Twenty patients (29.4%) received a follow-up thyroid ultrasound, eleven (16.2%) of whom underwent fine needle aspiration (FNA) for cytopathologic diagnosis. Among these, six (8.8%) underwent thyroid resection, with final pathology demonstrating papillary carcinoma in five (7.4%) and benign pathology in one. CONCLUSIONS: Despite the low incidence of thyroid nodules and low risk of thyroid malignancy in the general pediatric population, we found a significant rate of malignancy in CT-detected ITNs in our pediatric oncology patients, and recommend ultrasound and FNA of these nodules in this high-risk population. LEVEL OF EVIDENCE: Level IV, retrospective study with no comparison group.

A 20-year retrospective analysis of CT-based pre-operative identification of pulmonary metastases in patients with osteosarcoma: A single-center review.

PURPOSE: Cooperative studies support complete metastasectomy in osteosarcoma (OS). Pre-operative CT is used to identify and quantify metastases and can facilitate minimally invasive techniques. Here we assess the accuracy of pre-operative CT compared to findings at thoracotomy and its change over time. METHODS: We reviewed OS thoracotomies performed at our institution from 1996 to 2015. The number of metastases identified on pre-operative chest CT was compared to the number of metastases seen on pathology (both metastases with viable cells and non-viable, osteoid-only metastases). RESULTS: Eighty-eight patients underwent 161 thoracotomies with a median of 14days (range, 1-85) between CT and surgery, a median of 2 CT-identified lesions (range, 0-15), and a median of 4 resected lesions (range, 1-25). In 56 (34.8%) cases, more metastases were found surgically than were seen on CT, and among these, 34 (21.1%) had a greater number of viable metastases. There was poor overall correlation between CT and pathology findings (Kendall Tau-b=0.506), regardless of CT slice thickness, decade of thoracotomy, or total number of CT-identified lesions. CONCLUSIONS: CT accuracy in pre-operatively quantifying OS pulmonary metastases has not improved in recent decades. Consequently, we recommend an open technique with direct lung palpation for complete identification and resection of OS pulmonary metastases. LEVEL OF EVIDENCE: Level IV, retrospective study with no comparison group.

Paratesticular rhabdomyosarcoma: Importance of initial therapy.

PURPOSE: To evaluate factors associated with progression-free and disease-specific survival in patients with paratesticular rhabdomyosarcoma, we performed a cohort study. Also, since many patients present to our institution after initial therapy, we analyzed the effects of salvage therapy for scrotal violation. PATIENTS AND METHODS: We retrospectively reviewed the records of all consecutive patients with histologically confirmed paratesticular rhabdomyosarcoma treated at our institution between 1978 and 2015. Fifty-one patients were initially identified, but two with incomplete data were excluded from analysis. Variables evaluated for correlation with survival were TNM staging, Children's Oncology Group Soft Tissue Sarcoma pretreatment staging, margins at initial resection, presence of scrotal violation, hemiscrotectomy and/or scrotal radiation. The log-rank test was used to compare survival distributions. RESULTS: For the analytic cohort of 49 patients, the median age and follow-up were 15.7years (95% CI: 14.2-17.5, range: 0.8-25.1years) and 6.9years (95% CI: 4.4-9.0, range 0.2-37.5years), respectively. The 5-year overall disease-specific survival was 78.7% (95% CI: 67.7%-91.4%) and the progression-free survival was 66.9% (95% CI: 54.8%-81.6%). Median time to recurrence was 0.9years (95% CI: 0.7-0.9, range 0.1-6.2years). Scrotal violation occurred in 41% (n=20) and tripled the risk of recurrence for patients not appropriately treated with either hemiscrotectomy or scrotal radiation therapy (RR=3.0, 95% CI: 1.16-7.73). CONCLUSIONS: The strongest predictors of disease-specific survival were nodal status and distant metastasis at diagnosis. Scrotal violation remains a problem in paratesticular rhabdomyosarcoma and is a predictor of disease progression unless adequately treated. The risk of progression could be reduced with appropriate initial resection. LEVEL OF EVIDENCE: Level IV; retrospective study with no comparison group.

Scanning electrochemical microscopy of DNA monolayers modified with Nile Blue.

Scanning electrochemical microscopy (SECM) is used to probe long-range charge transport (CT) through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed at a specific location in the DNA film. At substrate potentials negative of the formal potential of covalently attached NB, the electrocatalytic reduction of Fe(CN)6(3-) generated at the SECM tip is observed only when NB is located at the DNA/solution interface; for DNA films containing NB in close proximity to the DNA/electrode interface, the electrocatalytic effect is absent. This behavior is consistent with both rapid DNA-mediated CT between the NB intercalator and the gold electrode as well as a rate-limiting electron transfer between NB and the solution phase Fe(CN)6(3-). The DNA-mediated nature of the catalytic cycle is confirmed through sequence-specific and localized detection of attomoles of TATA-binding protein, a transcription factor that severely distorts DNA upon binding. Importantly, the strategy outlined here is general and allows for the local investigation of the surface characteristics of DNA monolayers both in the absence and in the presence of DNA binding proteins. These experiments highlight the utility of DNA-modified electrodes as versatile platforms for SECM detection schemes that take advantage of CT mediated by the DNA base pair stack.