A novel treatment strategy utilizing panobinostat for high-risk and treatment-refractory hepatoblastoma.

BACKGROUND & AIMS: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To develop new therapeutic strategies for these patients, our laboratory has developed a preclinical testing pipeline. Given that histone deacetylase (HDAC) inhibition has been proposed for HB, we hypothesized that we could find an effective combination treatment strategy utilizing HDAC inhibition. METHODS: RNA sequencing, microarray, NanoString, and immunohistochemistry data of patient HB samples were analyzed for HDAC class expression. Patient-derived spheroids (PDSp) were used to screen combination chemotherapy with an HDAC inhibitor, panobinostat. Patient-derived xenograft (PDX) mouse models were developed and treated with the combination therapy that showed the highest efficacy in the PDSp drug screen. RESULTS: HDAC RNA and protein expression were elevated in HB tumors compared to normal livers. Panobinostat (IC50 of 0.013-0.059 µM) showed strong in vitro effects and was associated with lower cell viability than other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). All four models responded to VIP therapy with a decrease in tumor size compared to placebo. After 6 weeks of treatment, two models demonstrated necrotic cell death, with lower Ki67 expression, decreased serum alpha fetoprotein and reduced tumor burden compared to paired VI- and placebo-treated groups. CONCLUSIONS: Utilizing a preclinical HB pipeline, we demonstrate that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from patients with high-risk, relapsed, and treatment-refractory HB. IMPACT AND IMPLICATIONS: Patients with treatment-refractory hepatoblastoma have limited treatment options with survival rates of less than 50%. Our manuscript demonstrates that combination therapy with vincristine, irinotecan, and panobinostat reduces the size of high-risk, relapsed, and treatment-refractory tumors. With this work we provide preclinical evidence to support utilizing this combination therapy as an arm in future clinical trials.

Molecular Profiling of a Hepatocellular Neoplasm Not Otherwise Specified (HCN-NOS) Demonstrates Distinct Molecular Features in Hepatoblastoma and HCC-Like Components.

Hepatoblastomas (HB) are embryonal tumors with quiet genomes diagnosed mostly in children under 3 years of age and often cured by surgical resection and chemotherapy. However, a subset of HBs behave aggressively, displaying characteristic histologic features and higher genomic instability. Hepatocellular neoplasm-not otherwise specified (HCN-NOS) is a provisional diagnostic category for tumors exhibiting either intermediate or a combination of both HB and hepatocellular carcinoma (HCC) histological features. In this study, we characterized an HCN-NOS diagnosed in a 3-year-old patient presenting with a liver mass, in which both HB and HCC histological components were amendable to macro-dissection and molecular profiling. The spectrum of mutations, copy number changes, mRNA, and protein expression profiles within these 2 histologically distinct tumor areas demonstrate molecular heterogeneity and suggest intratumoral clonal evolution of this hepatocellular CTNNB1-mutant lesion.

Childhood and Adolescent Relapsed/Refractory Aggressive B-Cell Lymphomas With t(8;14) and BCL2 Expression, Burkitt Lymphoma Versus Diffuse Large B-Cell Lymphoma: A Diagnostic Challenge.

We present 2 diagnostically challenging cases of pediatric/adolescent relapsed/refractory aggressive mature B-cell non-Hodgkin lymphoma (B-NHL) within the spectrum of Burkitt lymphoma and diffuse large B-cell lymphoma and illustrate the different therapeutic regimens that are employed for pediatric and adult cancer centers. Both cases displayed varying-sized lymphoma cells with occasional single prominent nucleoli and heterogeneous BCL2 expression. Cytogenetics revealed complex karyotypes with t(8:14)(q24.2;q32) and IGH::MYC rearrangement by FISH. Next generation sequencing revealed deleterious TP53 and MYC mutations. We concluded that both could be diagnosed as "DLBCL-NOS with MYC rearrangement" using the current pathologic classifications, 2022 International Consensus Classification (ICC) and World Health Organization Classifications of Haematolymphoid Tumors (WHO-HAEM5). This report illustrates diagnostic challenges and treatment dilemmas that may be encountered, particularly for adolescent and young adults (AYA).

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.

Programmed cell death ligand 1 expression in aggressive pediatric non-Hodgkin lymphomas: frequency, genetic mechanisms, and clinical significance.

Programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are immunomodulatory molecules overexpressed in lymphomas and are promising immunotherapy targets for hematologic malignancies. However, studies of PD-1/PD-L1 overexpression and their clinical significance in aggressive pediatric non-Hodgkin lymphomas (NHL) are limited. We assessed PD-1/PD-L1 overexpression using immunohistochemistry in 68 aggressive pediatric NHL: ALK-positive anaplastic large cell lymphoma (ALK+ ALCL, n=8), Burkitt lymphoma (BL, n=27), and large B-cell lymphoma (LBCL) de novo LBCL, n=22 and diffuse LBCL arising as monomorphic post-transplant lymphoproliferative disorder [PTLD-DLBCL], n=11. In LBCL, correlations between PD-L1 overexpression and Epstein-Barr virus (EBV) status, cell of origin, stage, nodal status, overall survival (OS), and event-free survival (EFS) were examined. The genetic mechanisms of PD-L1 overexpression were investigated using targeted next-generation sequencing (NGS) and cytogenetic data. All ALK+ ALCL samples, 50.0% of de novo LBCL (11/22), 72.7% of PTLD-DLBCL (8/11), and no BL overexpressed PD-L1. Overexpressed PD-L1 correlated with EBV positivity (P=0.033) in LBCL and lower EFS in de novo LBCL (P=0.017). NGS of select LBCL revealed distinct somatic mutations and an ultra-hypermutated PTLD-DLBCL. Most cases with 9p24.1 copy gains overexpressed PD-L1 although some cases had no discernible genetic drivers of PD-L1 overexpression. Overexpressed PD-L1 is common in pediatric LBCL, associated with EBV positivity and 9p24.1 gains, and may have prognostic significance in de novo LBCL. Furthermore, diverse molecular mechanisms for PD-L1 overexpression in aggressive pediatric NHL can occur. Thus, additional studies exploring the therapeutic and prognostic significance and molecular mechanisms of PD-L1 overexpression in aggressive pediatric NHL are warranted.

Integrated DNA and RNA sequencing reveals targetable alterations in metastatic pediatric papillary thyroid carcinoma.

BACKGROUND: Pediatric papillary thyroid carcinoma (PTC) is clinically and biologically distinct from adult PTC. We sequenced a cohort of clinically annotated pediatric PTC cases enriched for high-risk tumors to identify genetic alterations of relevance for diagnosis and therapy. METHODS: Tumor DNA and RNA were extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation using a custom 124-gene hybridization capture panel and the 75-gene Archer Oncology Research Panel, respectively. NGS libraries were sequenced on an Illumina MiSeq. RESULTS: Thirty-six pediatric PTC cases were analyzed. Metastases were frequently observed to cervical lymph nodes (29/36, 81%), with pulmonary metastases less commonly found (10/36, 28%). Relapsed or refractory disease occurred in 18 patients (18/36, 50%). DNA sequencing revealed targetable mutations in 8 of 31 tumors tested (26%), most commonly BRAF p.V600E (n = 6). RNA sequencing identified targetable fusions in 13 of 25 tumors tested (52%): RET (n = 8), NTRK3 (n = 4), and BRAF. Mutually exclusive targetable alterations were discovered in 15 of the 20 tumors (75%) with both DNA and RNA analyzed. Fusion-positive PTC was associated with multifocal disease, higher tumor staging, and higher American Thyroid Association risk levels. Both BRAF V600E mutations and gene fusions were correlated with the presence of cervical metastases. CONCLUSIONS: Targetable alterations were identified in 75% of pediatric PTC cases with both DNA and RNA evaluated. Inclusion of RNA sequencing for detection of fusion genes is critical for evaluation of these tumors. Patients with fusion-positive tumors were more likely to have features of high-risk disease.

Ependymoma Presenting as a -Rim-Enhancing Lesion in the Brainstem.

INTRODUCTION: The posterior fossa is the most common intracranial location for pediatric ependymoma. While ependymoma usually arises from the ventricular lining of the fourth ventricle as a solid mass, it rarely originates from the brainstem. Grade II ependymomas also infrequently appear as a cavitary ring-enhancing lesion. CASE PRESENTATION: We describe a case of a 6-year-old boy with an ependymoma arising within the medulla with imaging features of a thick-walled rim-enhancing cavitary lesion. A stereotactic biopsy was obtained which confirmed a grade II ependymoma. The patient received focal proton beam radiation therapy and is doing well with no concerns for disease progression at 28 months after diagnosis. CONCLUSION: Posterior fossa ependymomas typically arise from ependymal cells within the fourth ventricle or foramina of Luschka. They rarely invade or arise within the brainstem parenchyma. Our case had atypical imaging findings in addition to the atypical tumor location. The lesion was described as a thick-walled rim-enhancing focal cystic necrotic lesion centered within the medulla with surrounding nonenhancing expansile infiltrative changes. Ring-enhancing lesions can be seen in patients with anaplastic ependymoma, but is not commonly reported in grade II ependymomas. In summary, this report highlights a unique case of a posterior fossa ependymoma in a pediatric patient arising in an atypical brainstem location as well as having unique imaging features.

Pediatric myeloid sarcoma: a single institution clinicopathologic and molecular analysis.

Myeloid sarcoma (MS) is a neoplastic condition composed of immature myeloid cells involving an extramedullary site. We investigated underlying chromosomal and molecular alterations to assess potential molecular markers of prognosis and outcome in this rare pediatric disease. We conducted a retrospective review of clinicopathologic and cytogenetic data from 33 pediatric patients with MS (ages 1 month-18 years) at our institution over a 32 year period (1984-2016). Tissue-based cancer microarray and targeted next-generation sequencing analysis were performed on six cases. The median age at diagnosis was 2.8 years with a male-to-female ratio of 2.6:1. MS is commonly presented with concomitant marrow involvement (n = 12, 36.4%) or as a recurrence of acute myeloid leukemia (AML; n = 14, 42.4%). The skin (n = 18, 54.5%) and soft tissue (n = 9, 27.3%) were the most common sites of involvement. Twenty-one of 25 samples (84.0%) harbored chromosomal aberrations; KMT2A alterations (n = 10, 40.0%) or complex cytogenetics (n = 7, 28.0%) were most frequent. Mutations in RAS, tyrosine kinase, cell signaling, and chromatin remodeling genes were detected. When compared to pediatric patients with AML without extramedullary involvement (EMI), inferior overall survival (OS) was observed (18.8 months vs. 89.3 months, p = .008). Pediatric patients with MS with non-favorable cytogenetics [abnormalities other than t(8;21), inv(16)/t(16;16), or t(15;17)] had a significantly lower OS compared to patients with AML with non-favorable cytogenetics and no extramedullary involvement (8.0 months vs. 28.1 months, p < .001). Pediatric MS is a rare disease with diverse clinical presentations. Non-favorable cytogenetics may be a poor prognostic marker for pediatric patients with MS and molecular diagnostics can assist with risk stratification and identify potentially actionable targets.

Acquisition of Cholangiocarcinoma Traits during Advanced Hepatocellular Carcinoma Development in Mice.

Past studies have identified hepatic tumors with mixed hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) characteristics that have a more aggressive behavior and a poorer prognosis than classic HCC. Whether this pathologic heterogeneity is due to a cell of origin of bipotent liver progenitors or the plasticity of cellular constituents comprising these tumors remains debated. In this study, we investigated the potential acquisition of CC-like traits during advanced development of HCC in mice. Primary and rare high-grade HCC developed in a genetic mouse model. A mouse model of highly efficient HCC invasion and metastasis by orthotopic transplantation of liver cancer organoids propagated from primary tumors in the genetic model was further developed. Invasive/metastatic tumors developed in both models closely recapitulated advanced human HCC and displayed a striking acquisition of CC-related pathologic and molecular features, which was absent in the primary HCC tumors. Our study directly demonstrates the pathologic evolution of HCC during advanced tumor development, providing the first evidence that tumors with mixed HCC and CC features, or at least a subset of these tumors, represent a more advanced developmental stage of HCC. Finally, liver cancer organoid-generated high-grade tumors exhibited significantly increased extracellular vesicle secretion, suggesting that identifying tumor-specific extracellular vesicle proteins in plasma may be a promising tool for liver cancer detection.

Sustained remission with azacitidine monotherapy and an aberrant precursor B-lymphoblast population in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) has a poor prognosis in general, with hematopoietic stem cell transplant (HSCT) remaining the standard of care for cure. The hypomethylating agent, azacitidine, has been used as a bridging therapy to transplant. However, no patients have been treated with azacitidine without an HSCT post azacitidine. We report on an infant with JMML with somatic KRAS G12A mutation and monosomy 7 who achieved sustained remission following azacitidine monotherapy. He also developed an aberrant B-lymphoblast population which declined with similar kinetics as his JMML-associated abnormalities, suggesting that a B-lymphoblast population in JMML does not always progress to acute leukemia.

The use of BRAF V600E mutation-specific immunohistochemistry in pediatric Langerhans cell histiocytosis.

BRAF p.V600E mutations are detected in greater than 50% of pediatric Langerhans cell histiocytosis (LCH) lesions. However, the use of mutation-specific BRAF V600E immunohistochemistry (IHC) as a surrogate for molecular testing in pediatric LCH is unknown. We tested the mutation-specific BRAF V600E monoclonal antibody (clone VE1) in formalin-fixed, paraffin-embedded LCH samples from 26 pediatric patients (14 males and 12 females, ages 7 mo-17 y) using allele-specific real-time polymerase chain reaction (PCR) with a limit of detection of 0.5% as the comparative gold standard. BRAF VE1 staining was scored for both intensity (0-3+) and percentage of immunoreactive tumor cells (0%-100%). BRAF VE1 immunoreactivity was determined using both lenient (≥1+, ≥1%) and stringent (≥2+, ≥10%) scoring criteria. Using lenient-scoring criteria, we found that the sensitivity and specificity of IHC compared with allele-specific real-time PCR were 100.0% and 18.2%, respectively. The poor specificity of lenient IHC analysis was attributable to weak, 1+ staining in both BRAF-mutated and wild-type LCH. Using stringent-scoring criteria, we found that specificity improved to 100.0% at the expense of sensitivity that decreased to 80.0%. Stringent scoring generated 3 false-negative results, but in all cases, neoplastic tissue comprised less than 5% of the stained section and/or the specimen was decalcified. In conclusion, highly sensitive molecular assays remain the gold standard for BRAF mutation analysis in LCH paraffin-embedded lesions. To avoid false-positive results, unequivocal VE1 staining of 2+ intensity in greater than or equal to 10% neoplastic histiocytes is required. However, negative VE1 results require additional studies to exclude false-negatives, and stringent-scoring criteria may not be optimal for scant or decalcified specimens.

USP6 activation in nodular fasciitis by promoter-swapping gene fusions.

Nodular fasciitis is a self-limited myofibroblastic lesion that can be misdiagnosed as a sarcoma as a result of its rapid growth, cellularity, and sometimes prominent mitotic activity. A recurrent translocation t(17;22) has been identified in nodular fasciitis, fusing the coding region of USP6 to the promoter region of MYH9, and resulting in increased USP6 expression. A subset of cases show USP6 rearrangement without the typical fusion variants by RT-PCR, or any MYH9 rearrangement by FISH. We sought to further characterize such tumors using molecular diagnostic assays. A novel RT-PCR assay was designed to detect the two known MYH9-USP6 fusion types in formalin-fixed paraffin-embedded and frozen tissue, and a break-apart FISH assay was designed to detect USP6 rearrangement. Twenty-six cases of nodular fasciitis diagnosed between 2002 and 2013 were retrieved from the pathology files of our institutions and were confirmed to be positive by FISH and/or RT-PCR. Seven samples showed USP6 rearrangement by FISH but were negative for MYH9-USP6 fusion by RT-PCR; these cases were subjected to a next-generation sequencing assay utilizing anchored multiplex PCR technology. This assay targets a single partner gene associated with fusions in bone and soft tissue tumors for agnostic detection of gene fusion partners. Novel fusion partners were identified in all seven cases and confirmed by RT-PCR. Structurally, all fusions consisted of the juxtaposition of the entire coding region of USP6 with the promoter of the partner gene, driving increased USP6 expression. This study confirms the neoplastic nature of nodular fasciitis, defines additional pathogenic fusion partners, and adds to the growing body of literature on USP6-associated neoplasia. Given the diagnostic challenges of these tumors, molecular assays can be useful ancillary tools; however, the prevalence of promoter swapping must be recognized when interpreting results.

Mucoepidermoid Carcinoma in Children: A Single Institutional Experience.

PURPOSE AND OBJECTIVE: To determine the clinicopathologic and molecular features and outcome of children with mucoepidermoid carcinoma (MEC). METHODS: A retrospective analysis of clinical and histopathologic findings was performed in patients with MEC diagnosed at Texas Children's Cancer Center between 2000 and 2014. RESULTS: Ten female and four male patients with median age 12 years (range 7-19 years) were included in the study. Tumors involved major salivary glands, minor salivary glands of the palate, and the tracheobronchial tree. Nine of 11 patients with salivary MEC underwent more than one surgical resection at the time of initial diagnosis to achieve a gross total resection. Three patients with tracheobronchial tumors underwent pulmonary lobectomy. Three patients received postoperative radiation therapy. No patient was treated with chemotherapy. Histopathologic grades were classified as low (n = 2), intermediate (n = 9), and high (n = 3). All 12 patients with tumor tissue available for testing were positive for MECT1/MAML2 fusion transcripts. There were no deaths, metastases, or recurrences in this series, with a median follow-up of 24 months (range 5-96 months). CONCLUSIONS: Low to intermediate histopathologic grade MECs are more common than high grade MEC in children. In contrast to adults, MECT1/MAML2 fusion transcripts occur with a frequency of 100% in our pediatric MEC series. Complete excision is the treatment of choice and is associated with excellent outcome. The role of radiotherapy is unclear, but may be indicated in patients with high grade tumors with positive surgical margins.

Cutaneous nodular fasciitis with genetic analysis: a case series.

Nodular fasciitis is a benign self-limited myofibroblastic neoplasm, which usually involves the upper extremities and trunk of young patients. These tumors have been shown to harbor a translocation involving the MYH9 and USP6 genes, leading to overexpression of the latter. We report seven cases of nodular fasciitis with cutaneous presentations. All cases involved the dermis, with six involving the superficial subcutis, and one auricular tumor extending into cartilage. All cases showed USP6 rearrangement by fluorescence in situ hybridization; in two of three cases, the characteristic MYH9-USP6 fusion was shown by RT-PCR. All patients underwent conservative resection. Nodular fasciitis is an uncommon mesenchymal neoplasm that can occasionally present in superficial locations and is sometimes mistaken for a malignant process. Molecular testing can be useful to distinguish this entity from other cutaneous spindle cell tumors.

Pediatric Hepatocellular Adenomas: What Is Known and What Is New?

Current understanding and classification of pediatric hepatocellular adenomas (HCA) are largely based on adult data. HCAs are rare in children and, unlike in adults, are often seen in the context of syndromes or abnormal background liver. Attempts to apply the adult classification to pediatric tumors have led to several "unclassifiable" lesions. Although typically considered benign, few can show atypical features and those with beta-catenin mutations have a risk for malignant transformation. Small lesions can be monitored while larger (>5.0 cm) lesions are excised due to symptoms or risk of bleeding/rupture, etc. Management depends on gender, age, underlying liver disease, multifocality, size of lesion, histologic subtype and presence of mutation, if any. In this review, we summarize the data on pediatric HCAs and highlight our experience with their diagnosis and management.

Hereditary pulmonary arterial hypertension burden in pediatrics: A single referral center experience.

Introduction: Hereditary pulmonary arterial hypertension (HPAH) is a rare yet serious type of pulmonary arterial hypertension (PAH). The burden in the pediatric population remains high yet underreported. The objective of this study is to describe the distribution of mutations found on targeted PAH panel testing at a large pediatric referral center. Methods: Children with PAH panel administered by the John Welsh Cardiovascular Diagnostic Laboratory at Texas Children's Hospital and Baylor College of Medicine in Houston, Texas between October 2012 to August 2021 were included into this study. Medical records were retrospectively reviewed for clinical correlation. Results: Sixty-six children with PAH underwent PAH genetic testing. Among those, 9 (14%) children were found to have pathogenic mutations, 16 (24%) children with variant of unknown significance and 41 (62%) children with polymorphism (classified as likely benign and benign). BMPR2 mutation was the most common pathogenic mutation, seen in 6 of the 9 children with detected mutations. Hemodynamic studies showed higher pulmonary vascular resistance among those with pathogenic mutations than those without (17.4 vs. 4.6 Wood units). All children with pathogenic mutations had severe PAH requiring triple therapy. There were tendencies for higher lung transplantation rate but lower mortality among those with pathogenic mutations. Conclusions: Abnormalities on genetic testing are not uncommon among children with PAH, although majority are of unclear significance. However, children with pathogenic mutations tended to present with more severe PAH requiring aggressive medical and surgical therapies. Genetic testing should be routinely considered due to consequences for treatment and prognostic implications. Larger scale population studies and registries are warranted to characterize the burden of HPAH in the pediatric population specifically.

RNA Sequencing for Solid Tumor Fusion Gene Detection: Proficiency Testing Practice and Performance Comparison.

CONTEXT.­: Next-generation sequencing-based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. OBJECTIVE.­: To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). DESIGN.­: CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. RESULTS.­: Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. CONCLUSIONS.­: These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics.

Navigating relapsed hepatoblastoma: Predictive factors and surgical treatment strategy.

OBJECTIVE: Hepatoblastoma (HB) is the most common primary hepatic malignancy in childhood. Relapse occurs in more than 50% of high-risk patients with a high mortality due to ineffective salvage therapies. The purpose of this study is to identify risk factors for relapsed HB and predictors of survival in a single tertiary referral center. METHODS: A retrospective chart review showed 129 surgically treated HB patients from October 2004 to July 2020. Of the cohort, 22 patients presented with relapsed HB. Relapse was defined as re-appearance of malignancy after 4 weeks of normalized AFP and disappearance of all tumors on imaging. RESULTS: Patients with relapsed HB had a 5-year overall survival (OS) of 45.4% compared to 93.1% in those without relapse (p = 0.001). When comparing PRETEXT IV, microvascular invasion, metastatic disease, and age on multivariate logistic regression, only PRETEXT IV was an independent risk factor for relapsed HB with an OR of 2.39 (95% CI: 1.16-4.96; p = 0.019). Mixed epithelial and mesenchymal HB (12/19, 63.2%) was the most common histology of primary tumors while pure epithelial HB (13/15, 86.6%) was the most common relapsed histology. Combination of surgical and medical therapy for relapsed disease was predictive of survival with an HR of 16.3 (95% CI: 1.783-149.091; p = 0.013) compared to only chemotherapy. CONCLUSIONS: This study demonstrates that PRETEXT IV staging is an independent predictor of relapsed disease. The most common relapsed histology was epithelial, suggesting a potential selection or resistance of this component. Surgical resection is a critical component of multimodal therapy for relapsed HB.

A Validation Framework for Somatic Copy Number Detection in Targeted Sequencing Panels.

Somatic copy number alterations (SCNAs) in tumors are clinically significant diagnostic, prognostic, and predictive biomarkers. SCNA detection from targeted next-generation sequencing panels is increasingly common in clinical practice; however, detailed descriptions of optimization and validation of SCNA pipelines for small targeted panels are limited. This study describes the validation and implementation of a tumor-only SCNA pipeline using CNVkit, augmented with custom modules and optimized for clinical implementation by testing reference materials and clinical tumor samples with different classes of copy number variation (CNV; amplification, single copy loss, and biallelic loss). Using wet-bench and in silico methods, various parameters impacting CNV calling, including assay-intrinsic variables (establishment of normal reference and sequencing coverage), sample-intrinsic variables (tumor purity and sample quality), and CNV algorithm-intrinsic variables (bin size), were optimized. The pipeline was trained and tested on an optimization cohort and validated using an independent cohort with a sensitivity and specificity of 100% and 93%, respectively. Using custom modules, intragenic CNVs with breakpoints within tumor suppressor genes were uncovered. Using the validated pipeline, re-analysis of 28 pediatric solid tumors that had been previously profiled for mutations identified SCNAs in 86% (24/28) samples, with 46% (13/28) samples harboring findings of potential clinical relevance. Our report highlights the importance of rigorous establishment of performance characteristics of SCNA pipelines and presents a detailed validation framework for optimal SCNA detection in targeted sequencing panels.

Clinical Laboratory Testing Practices in Diffuse Gliomas Prior to Publication of 2021 World Health Organization Classification of Central Nervous System Tumors.

CONTEXT.­: Integration of molecular data into glioma classification supports diagnostic, prognostic, and therapeutic decision-making; however, testing practices for these informative biomarkers in clinical laboratories remain unclear. OBJECTIVE.­: To examine the prevalence of molecular testing for clinically relevant biomarkers in adult and pediatric gliomas through review of a College of American Pathologists proficiency testing survey prior to the release of the 2021 World Health Organization Classification of Central Nervous System Tumors. DESIGN.­: College of American Pathologists proficiency testing 2020 survey results from 96 laboratories performing molecular testing for diffuse gliomas were used to determine the use of testing for molecular biomarkers in gliomas. RESULTS.­: The data provide perspective into the testing practices for diffuse gliomas from a broad group of clinical laboratories in 2020. More than 98% of participating laboratories perform testing for glioma biomarkers recognized as diagnostic for specific subtypes, including IDH. More than 60% of laboratories also use molecular markers to differentiate between astrocytic and oligodendroglial lineage tumors, with some laboratories providing more comprehensive analyses, including prognostic biomarkers, such as CDKN2A/B homozygous deletions. Almost all laboratories test for MGMT promoter methylation to identify patients with an increased likelihood of responding to temozolomide. CONCLUSIONS.­: These findings highlight the state of molecular testing in 2020 for the diagnosis and classification of diffuse gliomas at large academic medical centers. The findings show that comprehensive molecular testing is not universal across clinical laboratories and highlight the gaps between laboratory practices in 2020 and the recommendations in the 2021 World Health Organization Classification of Central Nervous System Tumors.