Stephen F. Sarabia

BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children.

The BCOR–CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative ‘Ewing-like’ sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired-end RNA sequencing (RNA-seq) we unexpectedly identified BCOR–CCNB3 fusion transcripts in an undifferentiated spindle-cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR–CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR–CCNB3 sarcoma were males diagnosed in mid childhood (7–13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all six cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR–CCNB3 fusion-positive sarcomas, a newly emerging entity within the undifferentiated unclassified sarcoma category and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors.

Genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups

Despite being the most common liver cancer in children, hepatoblastoma (HB) is a rare neoplasm. Consequently, few pretreatment tumors have been molecularly profiled, and there are no validated prognostic or therapeutic biomarkers for HB patients. We report on the first large‐scale effort to profile pretreatment HBs at diagnosis. Our analysis of 88 clinically annotated HBs revealed three risk‐stratifying molecular subtypes that are characterized by differential activation of hepatic progenitor cell markers and metabolic pathways: high‐risk tumors were characterized by up‐regulated nuclear factor, erythroid 2–like 2 activity; high lin‐28 homolog B, high mobility group AT‐hook 2, spalt‐like transcription factor 4, and alpha‐fetoprotein expression; and high coordinated expression of oncofetal proteins and stem‐cell markers, while low‐risk tumors had low lin‐28 homolog B and lethal‐7 expression and high hepatic nuclear factor 1 alpha activity. Conclusion: Analysis of immunohistochemical assays using antibodies targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis; our results pave the way for clinical collaborative studies to validate candidate biomarkers and test their potential to improve outcome for HB patients. (Hepatology 2017;65:104‐121).

Mucoepidermoid Carcinoma in Children: A Single Institutional Experience

To determine the clinicopathologic and molecular features and outcome of children with mucoepidermoid carcinoma (MEC).

Mucoepidermoid Carcinoma in Children

To determine the clinicopathologic and molecular features and outcome of children with mucoepidermoid carcinoma (MEC).

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.

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.

A Novel Cell Line Based Orthotopic Xenograft Mouse Model That Recapitulates Human Hepatoblastoma

Currently, preclinical testing of therapies for hepatoblastoma (HB) is limited to subcutaneous and intrasplenic xenograft models that do not recapitulate the hepatic tumors seen in patients. We hypothesized that injection of HB cell lines into the livers of mice would result in liver tumors that resemble their clinical counterparts. HepG2 and Huh-6 HB cell lines were injected, and tumor growth was monitored with bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Levels of human α-fetoprotein (AFP) were monitored in the serum of animals. Immunohistochemical and gene expression analyses were also completed on xenograft tumor samples. BLI signal indicative of tumor growth was seen in 55% of HepG2- and Huh-6-injected animals after a period of four to seven weeks. Increased AFP levels correlated with tumor growth. MRI showed large intrahepatic tumors with active neovascularization. HepG2 and Huh-6 xenografts showed expression of β-catenin, AFP, and Glypican-3 (GPC3). HepG2 samples displayed a consistent gene expression profile most similar to human HB tumors. Intrahepatic injection of HB cell lines leads to liver tumors in mice with growth patterns and biologic, histologic, and genetic features similar to human HB tumors. This orthotopic xenograft mouse model will enable clinically relevant testing of novel agents for HB.

Abstract B29: MDM4 is a major p53 regulator in hepatoblastoma

Purpose: Unresectable or metastatic hepatoblastoma generally confers a poor survival. MDM4 and MDM2 are the major negative regulators of p53, which has a predominantly wild type status in hepatoblastoma. Having previously shown that MDM4 has copy gain/amplification associated with the 1q32.1 amplicon in hepatoblastoma, we hypothesize that MDM4 is the predominant negative regulator of p53 function in hepatoblastoma and that blocking MDM4 will cause tumor cell death due to uninhibited p53 tumor suppressor activity. Methods: An MTT assay was used to measure NSC207895 (MDM4 inhibitor) and Nutlin-3a (MDM2 inhibitor) cytotoxicity on hepatoblastoma cell lines (HepG2, HepT1, and Huh-6) as well as a patient-derived hepatoblastoma cell line (PDCL-1). CCK-8 was used to assess effects of low dose NSC207895 on HB cell line proliferation. Soft agar was used to assess the effect of NSC207895 on hepatoblastoma colony formation. Immunoblotting and quantitative RT-PCR was used to measure expression of p53 downstream targets in hepatoblastoma after treatment with NSC207895. P53 knockdowns were created to assess the effect of NSC207895 in hepatoblastoma in the absence of p53. Cell cycle analysis was performed using BRDU. Results: Huh-6, HepG2, and HepT1 were all tested with Nutlin-3a and did not show significant cell death (IC50 >10μΜ) inhibition with NSC207895 caused significant cell death in Huh-6 (IC50=1.27μΜ), HepG2 (1.62μΜ), and HepT1 (2.05μΜ). A decrease in cell proliferation was noted with concentrations of NSC207895 as low as 0.05μΜ, 0.1μΜ and 0.3μΜ in HepG2, HepT1, and Huh-6 respectively. An increase in IC50 was seen in the p53 knockdowns compared to each respective control (HepG2: 12.6μΜ vs 3.38μΜ; HepT1: >50μΜ vs 1.45μΜ; Huh-6: 20.2μΜ vs 7.52μΜ). Increased PARP cleavage products and decreased MDM4 expression were seen on immunoblotting after treatment with NSC207895. By both immunoblotting and qPCR, the p53 downstream transcriptional targets, p21, BAX and PUMA, were increased after exposure to NSC207895 in all three cell lines. Cell cycle analysis showed an increased percentage of cells in G1/G0-phase and decreased percentage of actively replicating S-phase cells in HepG2 with NSC207895 treatment. Conclusion: Our data supports the hypothesis that in hepatoblastoma, MDM4 may be a dominant inhibitor of p53 and inhibition of MDM4 causes p53-mediated cell death and growth inhibition. Therefore, inhibition of MDM4 may be a viable drug target for treating hepatoblastoma. Citation Format: Yan Shi, Roma Patel, Emporia Hollingsworth, Stephen Sarabia, Jingling Jin, Dolores Lopez-Terrada, Sanjeev Vasudevan. MDM4 is a major p53 regulator in hepatoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B29.

Abstract 4267: Integrated genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups

Hepatoblastoma (HB), the most common primary liver cancer in children, is a clinically heterogeneous embryonal malignancy. Previous studies of HB have revealed a paucity of genetic alterations in these tumors, other than mutations activating the WNT signaling pathway, and have not provided an adequate biological explanation for the diverse clinical outcomes of children with HB. To identify critical genes and pathways in the pathogenesis of HB and provide insight into the biologic basis and clinical heterogeneity of these tumors, we performed an integrated genomic analysis of a large cohort of clinically-annotated tumor-normal pairs utilizing whole exome sequencing (n = 35), mRNA and miRNA expression arrays (n = 51; n = 58), and high-resolution copy number arrays (n = 47). Somatic mutations/deletions of CTNNB1 or rare germline mutations of APC were identified in >90% of cases, confirming the central role of the WNT pathway in HB pathogenesis. The only other gene targeted by recurrent mutations in our cohort was the transcription factor NFE2L2 (NRF2), which was found to harbor hotspot mutations at a similar frequency (5-10%) to that reported for hepatocellular carcinoma. Expression profiling revealed near-universal WNT-pathway activation: 7 of the 20 most highly expressed genes in HB were WNT-pathway related genes, and the WNT-pathway marker DKK1 was expressed hundreds of standard deviations above its levels in normal liver. Unsupervised hierarchical clustering identified three distinct molecular HB clusters that were characterized by differential activation of hepatic progenitor cell and metabolic pathways. Analysis of the expression and/or inferred-activity of transcription factors and highly expressed genes identified prognostic biomarkers, including the significantly predictive regulons of NFE2L2, LIN28b, HNF1A, and NOTCH1. These were used to identify clinical groups that overlapped with HB expression clusters and were associated with patient survival. Tumors in the high-risk group were characterized by high NFE2L2, low NOTCH1 activity, and high LIN28b expression and activity (corresponding to low let-7b expression), as well as high expression of the onco-fetal proteins AFP and GPC3 and stem cell markers EPCAM, DLK1, and SALL4. In contrast, the low-risk group was characterized by high HNF1a and NOTCH1 activities and low LIN28 expression and activity. Rare chromosomal gains and losses were characteristic of the low risk group of tumors, while gains of 20q13 (SALL4) and 1q chromosomal regions were primarily found in the high and intermediate risk groups. Immunohistochemistry for LIN28b, NFE2L2, HNF1alpha, PTEN, and EPCAM is being performed on a validation set of tumors to assess their reliability to classify formalin-fixed tumor specimens. Prospective studies will test the prognostic utility of this method for children with HB. Citation Format: Dolores H. Lopez-Terrada, Pavel Sumazin, Yidong Chen, Lisa Trevino, Stephen Sarabia, Oliver Hampton, Kayuri Patel, Toni-Ann Mistretta, Barry Zorman, Sarah Comerford, David Wheeler, Murali Chintagumpala, Rebecka M. Meyers, Milton J. Finegold, Gail Tomlinson, Donald W. Parsons. Integrated genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2015-4267

Abstract B79: Detectable BRAF-V600E mutation in circulating peripheral blood of patients with Langerhans cell histiocytosis correlates with risk organ involvement and residual disease

Purpose: Langerhans Cell Histiocytosis (LCH) is a clonal disorder characterized by inflammatory lesions with characteristic CD207+ dendritic cells (DCs). LCH has variable clinical presentations ranging from single lesions to potentially fatal multi-system “risk organ” disease. The etiology of LCH remains elusive, with debate of LCH as an inflammatory versus malignant disorder unresolved. The first recurrent somatic genetic mutation in LCH, BRAF-V600E, was recently reported in 57% of LCH lesions (Badalian-Very et al., 2010). Here we investigate the clinical significance of BRAF-V600E as a potential biomarker of risk organ or refractory disease. Methods: Formalin-fixed, paraffin embedded (FFPE) tissue, peripheral blood, and sorted peripheral monocyte/dendritic cell populations were genotyped for BRAF-V600E mutations with allele-specific, real-time PCR assays. The presence of BRAF-V600E mutations was correlated with clinical variables and analyzed with standard statistical methods. A subsequent validation set of 8 patient peripheral blood samples was identified for quantitative analysis of levels of BRAF-V600E positive cells with the BRAF Rotor-Gene Q (RGQ) PCR assay (Qiagen, Valencia, CA), and concordance with results from Qiagen qBiomarker qPCR assay was determined. Quantitation was performed using a delta Ct method of the BRAF-V600E assay, and results were reported as percentage of mutant cells in a background of wild-type cells using standard curves. Results: Lesions from 100 patients with LCH were genotyped, and 64% carried the V600E mutation, which localized to the infiltrating CD207+ DCs. In 16 patients with more than one lesion, BRAF status remained fixed, suggesting somatic mutation of BRAF is an early event. BRAF-V600E did not define specific clinical risk groups or impact overall survival, but it was associated with approximately two-fold higher risk of relapse (p=0.04). Furthermore, the cellular compartment carrying the mutation correlated with disease severity: the ability to detect BRAF-V600E in circulating mononuclear cells defined risk organ LCH with 100% sensitivity/87% specificity. The ability to detect BRAF-V600E in circulating blood cells in patients with risk organ LCH defined clinically detectable disease with 97% sensitivity/100% specificity. For development of a clinically reproducible minimal residual disease assay that would be CLIA-compliant and commercially available, a separate validation sample set was identified. With a limit of detection of 0.02% mutant cells in a background of wild-type cells, the RGQ assay correctly detected BRAF-V600E mutations in all 8 validation specimens and in known BRAF-V600E positive cell lines and did not detect mutations in 10 additional BRAF-V600E mutation negative clinical specimens (analytical specificity = 100%). The RGQ quantitative results correlated with the qBiomarker assay results (R2=0.924) with comparable analytical sensitivity. Conclusions: The molecular foothold of BRAF at the base of LCH pathogenesis will allow therapeutic strategies to move beyond empiric observation to risk-stratified and targeted approaches. Furthermore, effectiveness of therapy may be tested by following BRAF-V600E in peripheral blood cells as a marker of residual disease. Development of validated assays to test for BRAF-V600E in peripheral blood will assist in assigning risk status and assessing therapeutic response. Citation Format: Stephen J. Simko, Marie-Luise Berres, Karen Phaik-Har Lim, Tricia Peters, Jeremy Price, Philip J. Lupo, M. John Hicks, Albert Shih, Kenneth Heym, Kenneth L. McClain, Miriam Merad, Stephen Sarabia, Dolores Lopez-Terrada, Carl E. Allen. Detectable BRAF-V600E mutation in circulating peripheral blood of patients with Langerhans cell histiocytosis correlates with risk organ involvement and residual disease. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B79.