Histone H3 trimethylation on lysine 27 immunostaining pattern in DICER1-associated tumors.

Background: DICER1-associated tumors are heterogeneous and affect several organs. DICER1-associated primary intracranial sarcoma is associated with histone H3 trimethylation on lysine 27 (H3K27me3) loss in nucleus by immunohistochemistry. Methods: We explored the H3K27me3 immunostaining pattern in other DICER1-associated tumors. Twelve tumors from eleven patients with confirmed DICER1 mutations (sporadic and germline) data from a pancancer next-generation sequencing panel, and four tumors of pleuropulmonary blastoma (PPB) were retrieved from our database and stained with anti-H3K27me3 antibody. Results: The H3K27me3 expression in the nucleus showed heterogeneous mosaic loss in neoplastic Sertoli cell components in three of the five cases of moderately to poorly differentiated Sertoli-Leydig cell tumors. Among two tumors of DICER1-associated primary intracranial sarcoma, one showed complete loss of H3K27me3 in all neoplastic cells, whereas the other showed mosaic loss in the sarcomatous spindle cells. One DICER1-associated tumor with epithelial and mesenchymal differentiation, including pulmonary blastoma and PPB, showed mosaic loss of glandular epithelial and mesenchymal components. Four cases of type II PPB and a single case of type III PPB showed a similar mosaic loss of H3K27me3 staining restricted to large spindle cell components. All other components in all tumors-including Leydig cells; the areas of epithelial, cartilaginous, and rhabdomyomatous differentiation; and all cells of the remaining three cases (one papillary thyroid carcinoma and two cases of PPB type I)-demonstrated retained H3K27me3 staining. Conclusions: H3K27me3 expression is not universally lost in DICER1-associated tumors and thus is not predictive of DICER1 mutation status. The mosaic regional loss of H3K27me3 immunostaining is consistent in PPB type II and III, which can be a helpful diagnostic marker for these tumors and suggests a similarity to DICER1-associated intracranial sarcoma.

Comparative Clinicopathologic and Genomic Analysis of Hepatocellular Neoplasm, Not Otherwise Specified, and Hepatoblastoma.

Accurate diagnosis and treatment of hepatocellular neoplasm, not otherwise specified (HCN-NOS), poses significant challenges. Our study aimed to investigate the clinicopathologic and genomic similarities and differences between HCN-NOS and hepatoblastoma (HB) to guide diagnostic and treatment strategies. The clinicopathologic characteristics of 16 patients with HCN-NOS and 23 patients with HB were compared. Molecular studies, including the OncoKids DNA- and RNA-based next-generation sequencing panel, chromosomal microarray, and targeted Sanger sequencing analyses of CTNNB1 and TERT promoters, were employed. We found that patients with HCN-NOS were older (P < .001) and more frequently classified as high risk (P < .01), yet they showed no significant differences in alpha fetoprotein levels or survival outcomes compared with those with HB. HCN-NOS and HB had a comparable frequency of sequence variants, with CTNNB1 mutations being predominant in both groups. Notably, TERT promoter mutations (37.5%) and rare clinically significant variants (BRAF, NRAS, and KMT2D) were exclusive to HCN-NOS. HCN-NOS demonstrated a higher prevalence of gains in 1q, encompassing the MDM4 locus (17/17 vs 11/24; P < .001), as well as loss/loss of heterozygosity (LOH) of 1p (11/17 vs 6/24; P < .05) and chromosome 11 (7/17 vs 1/24; P < .01) when compared with HB. Furthermore, the recurrent loss/LOH of chromosomes 3, 4p, 9, 15q, and Y was only observed in HCN-NOS. However, no significant differences were noted in gains of chromosomes 2, 8, and 20, or loss/LOH of 4q and 11p between the 2 groups. Notably, no clinically significant gene fusions were detected in either group. In conclusion, our study reveals that HCN-NOS exhibits high-risk clinicopathologic features and greater structural complexity compared with HB. However, patients with HCN-NOS exhibit comparable alpha fetoprotein levels at diagnosis, CTNNB1 mutation rates, and survival outcomes when subjected to aggressive treatment, as compared with those with HB. These findings have the potential to enhance diagnostic accuracy and inform more effective treatments for HCN-NOS.

Pediatric Sertoli-Leydig Cell Tumors of the Ovary: An Integrated Study of Clinicopathological Features, Pan-cancer-Targeted Next-generation Sequencing and Chromosomal Microarray Analysis From a Single Institution.

Sertoli-Leydig cell tumors (SLCTs) are currently classified into 3 molecular subtypes: DICER1 -mutant (younger patient age), FOXL2 -mutant, and DICER1/FOXL2 -wildtype. However, it is not clear whether all pediatric SLCTs are DICER1 -mutant molecular subtypes and whether other molecular genetic aberrations besides DICER1 are involved in the pathogenesis and prognosis of these tumors. We studied comprehensive data for 8 cases of pediatric SLCTs, including clinicopathological features, pan-cancer-targeted next-generation sequencing/OncoKids panel, and chromosomal microarray analysis, to further analyze the correlation among clinicopathological features, molecular genetic aberrations, and prognosis. The ages of the patients ranged from 4 to 16 years (median, 14 y). Seven cases were moderately differentiated, and one was poorly differentiated with heterologous mesenchymal elements. Two cases had heterologous epithelium or retiform elements. Follow-up was available for all 8 patients (median, 49.5 mo). Seven patients were alive without evidence of recurrence or metastasis, and only case 5 developed metastases (synchronous bilateral pulmonary tumors with rhabdomyosarcomatous differentiation). All 8 tumors were found to harbor somatic hotspot DICER1 mutations, and 5 patients carried germline DICER1 mutations (2 of them had the phenotype of DICER1 syndrome). Together with recent studies, the DICER1 mutation frequency is 100% in pediatric SLCTs (n=27, age≤16 y). Copy number alterations were detected in 3 tumors; the only recurrent copy number alterations was the gain of whole chromosome 6 in case 5 and case 8. This is the first report describing clinicopathological features and molecular alterations in pediatric SLCTs. Our results demonstrate that all pediatric SLCTs belong to the DICER1 -mutant molecular subtype, highlighting that somatic hotspot DICER1 mutation detection has high sensitivity (100%) for the auxiliary diagnosis of pediatric SLCTs (age ≤16 y). Some pediatric SLCTs harbor molecular genetic aberrations other than DICER1 mutation, and their significance needs further study.

An Exome Capture-Based RNA-Sequencing Assay for Genome-Wide Identification and Prioritization of Clinically Important Fusions in Pediatric Tumors.

This study reports the development of an exome capture-based RNA-sequencing assay to detect recurring and novel fusions in hematologic, solid, and central nervous system tumors. The assay used Twist Comprehensive Exome capture with either fresh or formalin-fixed samples and a bioinformatic platform that provides fusion detection, prioritization, and downstream curation. A minimum of 50 million uniquely mapped reads, a consensus read alignment/fusion calling approach using four callers (Arriba, FusionCatcher, STAR-Fusion, and Dragen), and custom software were used to integrate, annotate, and rank the candidate fusion calls. In an evaluation of 50 samples, the number of calls varied substantially by caller, from a mean of 24.8 with STAR-Fusion to 259.6 with FusionCatcher; only 1.1% of calls were made by all four callers. Therefore a filtering and ranking algorithm was developed based on multiple criteria, including number of supporting reads, calling consensus, genes involved, and cross-reference against databases of known cancer-associated or likely false-positive fusions. This approach was highly effective in pinpointing known clinically relevant fusions, ranking them first in 47 of 50 samples (94%). Detection of pathogenic gene fusions in three diagnostically challenging cases highlights the importance of a genome-wide and nontargeted method for fusion detection in pediatric cancer.

Norepinephrine transporter and vesicular monoamine transporter 2 tumor expression as a predictor of response to 131 I-MIBG in patients with relapsed/refractory neuroblastoma.

BACKGROUND: Prior studies suggest that norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) mediate meta-iodobenzylguanidine (MIBG) uptake and retention in neuroblastoma tumors. We evaluated the relationship between NET and VMAT2 tumor expression and clinical response to 131 I-MIBG therapy in patients with neuroblastoma. METHODS: Immunohistochemistry (IHC) was used to evaluate NET and VMAT2 protein expression levels on archival tumor samples (obtained at diagnosis or relapse) from patients with relapsed or refractory neuroblastoma treated with 131 I-MIBG. A composite protein expression H-score was determined by multiplying a semi-quantitative intensity value (0-3+) by the percentage of tumor cells expressing the protein. RESULTS: Tumor samples and clinical data were available for 106 patients, of whom 28.3% had partial response (PR) or higher. NET H-score was not significantly associated with response (≥PR), though the percentage of tumor cells expressing NET was lower among responders (median 80% for ≥PR vs. 90% for

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. Highlights: Plasma membrane enriched proteomics defines surfaceome of neuroblastomaMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancersDLK1 expression is driven by a super-enhancer DLK1 silencing in neuroblastoma cells results in cellular differentiation ADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models.

Use of Surgical Adjuvants Does Not Decrease Recurrence of Aneurysmal Bone Cysts in Surgical Intervention With Pediatric Patients.

BACKGROUND: Aneurysmal bone cysts (ABCs) are benign active tumors often requiring intralesional curettage with or without adjuvants. The primary aim of this study was to analyze whether recurrence is influenced by the use of surgical adjuvants in pediatric patients with ABCs. Secondary aims examined recurrence rates based on age, sex, and physeal contact. METHODS: A retrospective review was performed at a tertiary pediatric hospital from 2004 to 2020. Inclusion criteria consisted of patients treated surgically for histologically confirmed ABCs with a minimum of 6 months follow-up. Patients with treatment for a recurrent tumor or incomplete records were excluded. Patient demographics, location of the lesion, treatment technique, and incidence of recurrence were collected. Statistical analyses were performed using STATA. RESULTS: There were 129 patients (74 males and 55 females) with a mean age of 11.5 ± 4.1 years and an average follow-up of 29.0 ± 25.4 months. The most common locations for ABCs were the femur and tibia. Of the patients, 53.5% had tumors abutting the physis, 28.7% had no physeal contact, and 17.8% had insufficient imaging to evaluate physeal contact. Surgical adjuvants (high-speed burr, coagulation, liquid nitrogen, and/or hydrogen peroxide) were used in 91 of the 129 cases (70.5%). There was no significant difference in recurrence when comparing those who received an adjuvant and those who did not (25.3% vs 23.7%, P = 1.000). Physeal contact was also not significantly associated with recurrence ( P = 0.146). Finally, patients younger than 6 years old were significantly more likely to have recurrence compared with those 6 years old or older (66.7% vs 21.7%, P = 0.007). CONCLUSION: Our study found no association between the use of surgical adjuvants and the risk of recurrence after intralesional curettage for ABCs. Although our study did demonstrate that patients 6 years old or younger had an increased rate of recurrence, no significant association was found regarding physeal contact or sex. These data indicate that surgical adjuvant may not affect the recurrence rates of pediatric patients with ABCs. LEVEL OF EVIDENCE: Level III. This retrospective review compares rates of recurrence based on the choice of surgical adjuvant.

Long-Term Outcomes in Patients With Localized Ewing Sarcoma Treated With Interval-Compressed Chemotherapy on Children's Oncology Group Study AEWS0031.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned coprimary or secondary analyses are not yet available. Clinical trial updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Long-term outcomes from Children's Oncology Group study AEWS0031 were assessed to determine whether the survival advantage of interval-compressed chemotherapy (ICC) was maintained over 10 years in patients with localized Ewing sarcoma (ES). AEWS0031 enrolled 568 eligible patients. Patients were randomly assigned to receive vincristine-doxorubicin-cyclophosphamide and ifosfamide-etoposide alternating once every 3 weeks (standard timing chemotherapy [STC]) versus once every 2 weeks (ICC). For this updated report, one patient was excluded because of uncertainty of original diagnosis. The 10-year event-free survival (EFS) was 70% with ICC compared with 61% with STC (P = .03), and 10-year overall survival (OS) was 76% with ICC compared with 69% with STC (P = .04). There was no difference in the 10-year cumulative incidence of second malignant neoplasms (SMNs; PC [see Data Supplement, online only] = .5). A test for interaction demonstrated that ICC provided greater risk reduction for patients with tumor volume ≥200 mL than for patients with tumors <200 mL, but no evidence for a significant interaction in other subgroups defined by age, primary site, and histologic response. With longer-term follow-up, ICC for localized ES is associated with superior EFS and OS without an increased risk for SMN compared with STC. ICC is associated with improved outcomes even in adverse-risk patient groups.

"Oncocytoid Renal Cell Carcinomas After Neuroblastoma" Represent TSC -mutated Eosinophilic Solid and Cystic Renal Cell Carcinomas : Association With Prior Childhood Malignancy and Multifocality With Therapeutic Implications.

The concept of oncocytoid renal cell carcinoma in patients who have survived neuroblastoma as a distinct biologic entity has been controversial since its original description in 1999. This is in part because similar oncocytoid renal cell carcinomas have been described in association with other pediatric cancers, and also because other renal cell carcinoma subtypes (such as MiT family translocation renal cell carcinoma) have been described in children who have survived neuroblastoma. We identified an index case of a child who survived medulloblastoma and developed multifocal bilateral oncocytoid renal cell carcinomas with morphology and immunophenotype compatible with eosinophilic solid and cystic renal cell carcinoma (ESC RCC) and demonstrated that both neoplasms harbored distinctive mutations in the TSC1/TSC2 genes. Remarkably, the child's remaining bilateral multifocal renal neoplasms completely responded to MTOR inhibitor therapy without need for further surgery. To confirm our hypothesis that oncocytoid renal cell carcinomas after childhood cancer represent ESC RCC, we obtained formalin-fixed paraffin-embedded tissue blocks from 2 previously published cases of oncocytoid renal cell carcinoma after neuroblastoma, confirmed that the morphology and immunophenotype was consistent with ESC RCC, and demonstrated that both cases harbored somatic TSC gene mutations. Both expressed markers previously associated with neoplasms harboring TSC gene mutations, glycoprotein nonmetastatic B, and cathepsin K. Of note, one of these patients had 2 ESC RCC which harbored distinctive TSC2 mutations, while the background kidney of the other patient had multiple small cysts lined by similar oncocytoid cells which showed loss of TSC2 protein. We then reviewed 3 of 4 cases from the original 1999 report of oncocytoid renal cell carcinomas after neuroblastoma, found that all 3 demonstrated morphology (including basophilic cytoplasmic stippling) that is characteristic of ESC RCC, showed that all 3 overexpressed glycoprotein nonmetastatic B, and showed that both cases with adequate material demonstrated loss of TSC2 protein and expressed cytokeratin 20 and cathepsin K by immunohistochemistry. In summary, "oncocytoid renal cell carcinomas after neuroblastoma" represent ESC RCC which are often multifocal in patients who have survived childhood cancer, likely representing an incompletely characterized tumor predisposition syndrome. MTOR-targeted therapy represents an effective therapeutic option for such patients to preserve functional nephrons.

Lorlatinib with or without chemotherapy in ALK-driven refractory/relapsed neuroblastoma: phase 1 trial results.

Neuroblastomas harbor ALK aberrations clinically resistant to crizotinib yet sensitive pre-clinically to the third-generation ALK inhibitor lorlatinib. We conducted a first-in-child study evaluating lorlatinib with and without chemotherapy in children and adults with relapsed or refractory ALK-driven neuroblastoma. The trial is ongoing, and we report here on three cohorts that have met pre-specified primary endpoints: lorlatinib as a single agent in children (12 months to <18 years); lorlatinib as a single agent in adults (≥18 years); and lorlatinib in combination with topotecan/cyclophosphamide in children (<18 years). Primary endpoints were safety, pharmacokinetics and recommended phase 2 dose (RP2D). Secondary endpoints were response rate and 123I-metaiodobenzylguanidine (MIBG) response. Lorlatinib was evaluated at 45-115 mg/m2/dose in children and 100-150 mg in adults. Common adverse events (AEs) were hypertriglyceridemia (90%), hypercholesterolemia (79%) and weight gain (87%). Neurobehavioral AEs occurred mainly in adults and resolved with dose hold/reduction. The RP2D of lorlatinib with and without chemotherapy in children was 115 mg/m2. The single-agent adult RP2D was 150 mg. The single-agent response rate (complete/partial/minor) for <18 years was 30%; for ≥18 years, 67%; and for chemotherapy combination in <18 years, 63%; and 13 of 27 (48%) responders achieved MIBG complete responses, supporting lorlatinib's rapid translation into active phase 3 trials for patients with newly diagnosed high-risk, ALK-driven neuroblastoma. ClinicalTrials.gov registration: NCT03107988 .

Fibroblasts and macrophages cooperate to create a pro-tumorigenic and immune resistant environment via activation of TGF-ß/IL-6 pathway in neuroblastoma.

Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF) and their precursor mesenchymal stromal cells (MSC) are often detected together in tumors, but how they cooperate is not well understood. Here, we show that TAM and CAF are the most abundant nonmalignant cells and are present together in untreated human neuroblastoma (NB) tumors that are also poorly infiltrated with T and natural killer (NK) cells. We then show that MSC and CAF-MSC harvested from NB tumors protected human monocytes (MN) from spontaneous apoptosis in an interleukin (IL)-6 dependent mechanism. The interactions of MN and MSC with NB cells resulted in a significant induction or increase in the expression of several pro-tumorigenic cytokines/chemokines (TGF-ß1, MCP-1, IL-6, IL-8, and IL-4) but not of anti-tumorigenic cytokines (TNF-α, IL-12) by MN or MSC, while also inducing cytokine expression in quiescent NB cells. We then identified a TGF-ß1/IL-6 pathway where TGF-ß1 stimulated the expression of IL-6 in NB cells and MSC, promoting TAM survival. Evidence for the contribution of TAM and MSC to the activation of this pathway was then provided in xenotransplanted NB tumors and patients with primary tumors by demonstrating a direct correlation between the presence of CAF and p-SMAD2 and p-STAT3. The data highlight a new mechanism of interaction between TAM and CAF supporting their pro-tumorigenic function in cancer.

Potential methylation-regulated genes and pathways in hepatocellular neoplasm, not otherwise specified.

Background and Aims: The molecular basis of hepatocellular neoplasm, not otherwise specified (HCN-NOS) is unknown. We aimed to identify gene expression patterns, potential methylation-regulated genes and pathways that characterize the tumor, and its possible relationship to hepatoblastoma and hepatocellular carcinoma (HCC). Approach & Results: Parallel genome-wide profiling of gene expression (RNAseq) and DNA methylation (EPIC850) was performed on 4 pairs of pre-treatment HCN-NOS tumors and adjacent non-tumor controls. 2530 significantly differentially expressed genes (DEGs) were identified between tumors and controls. Many of these DEGs were associated with hepatoblastoma and/or HCC. Analysis Match in Ingenuity Pathway Analysis determined that the gene expression profile of HCN-NOS was unique but significantly similar to that of both hepatoblastoma and HCC. A total of 27,195 CpG sites (CpGs) were significantly differentially methylated (DM) between tumors and controls, with a global hypomethylation pattern and predominant CpG island hypermethylation in promotor regions. Aberrant DNA methylation predominated in Developmental Process and Molecular Function Regulator pathways. Embryonic stem cell pathways were significantly enriched. In total, 1055 aberrantly methylated (at CpGs) and differentially expressed genes were identified, including 25 upstream regulators and sixty-one potential CpG island methylation-regulated genes. Eight methylation-regulated genes (TCF3, MYBL2, SRC, HMGA2, PPARGC1A, SLC22A1, COL2A1 and MYCN) had highly consistent gene expression patterns and prognostic value in patients with HCC, based on comparison to publicly available datasets. Conclusions: HCN-NOS has a unique, stem-cell like gene expression and DNA methylation profile related to both hepatoblastoma and HCC but distinct therefrom. Further, 8 methylation-regulated genes associated with prognosis in HCC were identified.

Preclinical Development of [211At]meta- astatobenzylguanidine ([211At]MABG) as an Alpha Particle Radiopharmaceutical Therapy for Neuroblastoma.

PURPOSE: [131I]meta-iodobenzylguanidine ([131I]MIBG) is a targeted radiotherapeutic administered systemically to deliver beta particle radiation in neuroblastoma. However, relapses in the bone marrow are common. [211At]meta-astatobenzylguanidine ([211At] MABG) is an alpha particle emitter with higher biological effectiveness and short path length which effectively sterilizes microscopic residual disease. Here we investigated the safety and antitumor activity [211At]MABG in preclinical models of neuroblastoma. EXPERIMENTAL DESIGN: We defined the maximum tolerated dose (MTD), biodistribution, and toxicity of [211At]MABG in immunodeficient mice in comparison with [131I]MIBG. We compared the antitumor efficacy of [211At]MABG with [131I]MIBG in three murine xenograft models. Finally, we explored the efficacy of [211At]MABG after tail vein xenografting designed to model disseminated neuroblastoma. RESULTS: The MTD of [211At]MABG was 66.7 MBq/kg (1.8 mCi/kg) in CB17SC scid-/- mice and 51.8 MBq/kg (1.4 mCi/kg) in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Biodistribution of [211At]MABG was similar to [131I]MIBG. Long-term toxicity studies on mice administered with doses up to 41.5 MBq/kg (1.12 mCi/kg) showed the radiotherapeutic to be well tolerated. Both 66.7 MBq/kg (1.8 mCi/kg) single dose and fractionated dosing 16.6 MBq/kg/fraction (0.45 mCi/kg) × 4 over 11 days induced marked tumor regression in two of the three models studied. Survival was significantly prolonged for mice treated with 12.9 MBq/kg/fraction (0.35 mCi/kg) × 4 doses over 11 days [211At]MABG in the disseminated disease (IMR-05NET/GFP/LUC) model (P = 0.003) suggesting eradication of microscopic disease. CONCLUSIONS: [211At]MABG has significant survival advantage in disseminated models of neuroblastoma. An alpha particle emitting radiopharmaceutical may be effective against microscopic disseminated disease, warranting clinical development.

Intraoperative Diagnosis for Pediatric Brain Tumors.

Central nervous system (CNS) tumors are now the most common type of solid tumor in individuals aged 0-19 years, with an incidence rate in the United States around 5 per 100,000, accounting for about 1 out of 4 childhood cancers. Pediatric pathologists encounter brain tumor cases with varying frequency, but many of these encounters begin in the context of intraoperative consultation or "frozen section." This review provides an overview of the technical aspects of intraoperative consultation specific to, or more helpful in, CNS tumors, emphasizing helpful cytologic and histologic features of the more commonly encountered pediatric CNS tumors, and illustrating some common diagnostic pitfalls and how these may be avoided.

PLAG1 Immunohistochemical Staining Is a Surrogate Marker for PLAG1 Fusions in Lipoblastomas.

BACKGROUND: The hallmark of lipoblastoma is a PLAG1 fusion. PLAG1 protein overexpression has been reported in sporadic PLAG1-rearranged lipoblastomas. METHODS: We evaluated the utility of PLAG1 immunohistochemical staining (IHC) in 34 pediatric lipomatous tumors, correlating the results with histology and conventional cytogenetics, FISH and/or next generation sequencing (NGS) results. RESULTS: The study included 24 lipoblastomas, divided into 2 groups designated as "Lipoblastoma 1" with both lipoblastoma histology and PLAG1 rearrangement (n = 16) and "Lipoblastoma 2" with lipoblastoma histology but without PLAG1 cytogenetic rearrangement (n = 8), and 10 lipomas with neither lipoblastoma histology nor a PLAG1 rearrangement. Using the presence of a fusion as the "gold standard" for diagnosing lipoblastoma (Lipoblastoma 1), the sensitivity of PLAG1 IHC was 94%. Using histologic features alone (Lipoblastoma 1 + 2), the sensitivity was 96%. Specificity, as defined by the ability to distinguish lipoma from lipoblastoma, was 100%, as there were no false positives in the lipoma group. CONCLUSIONS: Cytogenetics/molecular testing is expensive and may not be ideal for detecting PLAG1 fusions because PLAG1 fusions are often cytogenetically cryptic and NGS panels may not include all partner genes. PLAG1 IHC is an inexpensive surrogate marker of PLAG1 fusions and may be useful in distinguishing lipoblastomas from lipomas.

Primary Adrenal Malignant Rhabdoid Tumor in a 14-Year-Old Female: A Case Report and Literature Review.

Malignant rhabdoid tumor (MRT) is a rare, SWItch/sucrose nonfermentable-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1)-deficient, aggressive tumor, occurring predominantly in children below 3 years of age. Primary adrenal MRT is extremely rare, with only 3 cases reported in the literature. A previously healthy 14-year-old female presented with left upper quadrant/epigastric abdominal pain. Imaging studies revealed an 8.0 × 8.0 × 6.5 cm, heterogeneous, partially enhancing mass along the superior margin of the left kidney encasing the adrenal gland. Surgical resection of the tumor revealed a hypercellular heterogeneous neoplasm arising from the adrenal gland. It was composed predominantly of primitive small round blue cells with focal true rosettes and areas of vague glandular epithelial differentiation and chondroid differentiation. Classic rhabdoid-type cytoplasmic inclusions were focally present. Mitoses, tumor necrosis, and hemorrhage were readily seen. Tumor cells showed complete loss of SMARCB1 (INI1) nuclear staining, demonstrated strong, and diffuse positivity for glypican 3, patchy positivity for CD99, cytokeratin, Sal-like protein 4, Lin-28 homolog A, epithelial membrane antigen, and S100. Molecular studies revealed biallelic frameshift mutations in the SMARCB1 gene (c.673delG and c.683dupT) without pathogenic copy number aberrations. The histologic, immunohistochemical, and molecular findings support a diagnosis of MRT. The unusual age, location, and mutations of this case expand the clinicopathologic and molecular spectrum of MRT.

Phase III Trial Adding Vincristine-Topotecan-Cyclophosphamide to the Initial Treatment of Patients With Nonmetastatic Ewing Sarcoma: A Children's Oncology Group Report.

PURPOSE: The primary aim of this phase III randomized trial was to test whether the addition of vincristine, topotecan, and cyclophosphamide (VTC) to interval compressed chemotherapy improved survival outcomes for patients with previously untreated nonmetastatic Ewing sarcoma. METHODS: Patients were randomly assigned to receive standard five-drug interval compressed chemotherapy (regimen A) for 17 cycles or experimental therapy with five cycles of VTC within the 17 cycles (regimen B). Patients were stratified by age at diagnosis (< 18 years and ≥18 years) and tumor site (pelvic bone, nonpelvic bone, and extraosseous). Tumor volume at diagnosis was categorized as < 200 mL or ≥ 200 mL. Local control occurred following six cycles. Histologic response was categorized as no viable or any viable tumor. Event-free survival (EFS) and overall survival (OS) were compared between randomized groups with stratified log-rank tests. RESULTS: Of 642 enrolled patients, 309 eligible patients received standard and 320 received experimental therapy. The 5-year EFS and OS were 78% and 87%, respectively. There was no difference in survival outcomes between randomized groups (5-year EFS regimen A v regimen B, 78% v 79%; P = .192; 5-year OS 86% v 88%; P = .159). Age and primary site did not affect the risk of an EFS event. However, age ≥ 18 years was associated with an increased risk of death at 5 years (hazard ratio 1.84; 95% CI, 1.15 to 2.96; P = .009). The 5-year EFS rates for patients with pelvic, nonpelvic bone, and extraosseous primary tumors were 75%, 78%, and 85%, respectively. Tumor volume ≥ 200 mL was significantly associated with lower EFS. CONCLUSION: While VTC added to five-drug interval compressed chemotherapy did not improve survival, these outcomes represent the best survival estimates to date for patients with previously untreated nonmetastatic Ewing sarcoma.

A GPC2 antibody-drug conjugate is efficacious against neuroblastoma and small-cell lung cancer via binding a conformational epitope.

Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.

Image-Guided Biopsy for Relapsed Neuroblastoma: Focus on Safety, Adequacy for Genetic Sequencing, and Correlation of Tumor Cell Percent With Quantitative Lesion MIBG Uptake.

PURPOSE: Many novel therapies for relapsed and refractory neuroblastoma require tumor tissue for genomic sequencing. We analyze our experience with image-guided biopsy in these patients, focusing on safety, yield, adequacy for next-generation sequencing (NGS), and correlation of tumor cell percent (TC%) with quantitative uptake on 123I-meta-iodobenzylguanidine (MIBG) single-photon emission computed tomography with computed tomography (SPECT/CT). MATERIALS AND METHODS: An 11-year retrospective review of image-guided biopsy on 66 patients (30 female), with a median age of 8.7 years (range, 0.9-49 years), who underwent 95 biopsies (55 bone and 40 soft tissue) of relapsed or refractory neuroblastoma lesions was performed. RESULTS: There were seven minor complications (7%) and one major complication (1%). Neuroblastoma was detected in 88% of MIBG- or fluorodeoxyglucose-avid foci. The overall NGS adequacy was 69% (64% in bone and 74% in soft tissue, P = .37). NGS adequacy within neuroblastoma-positive biopsies was 88% (82% bone and 96% soft tissue, P = .11). NGS-adequate biopsies had a greater mean TC% than inadequates (51% v 18%, P = .03). NGS-adequate biopsies had a higher mean number of needle passes (7.5 v 3.4, P = .0002). The mean tissue volume from NGS-adequate soft-tissue lesions was 0.16 cm3 ± 0.12. Lesion:liver and lesion:psoas MIBG uptake ratios correlated with TC% (r = 0.74, r = 0.72, and n = 14). Mean TC% in NGS-adequate samples was 51%, corresponding to a lesion:liver ratio of 2.9 and a lesion:psoas ratio of 9.0. Thirty percent of biopsies showed an actionable ALK mutation or other therapeutically relevant variant. CONCLUSION: Image-guided biopsy for relapsed or refractory neuroblastoma was safe and likely to provide NGS data to guide therapy decisions. A lesion:liver MIBG uptake ratio of ≥ 3 or a lesion:psoas ratio of > 9 was associated with a TC% sufficient to deliver NGS results.

XPO1 inhibition with selinexor synergizes with proteasome inhibition in neuroblastoma by targeting nuclear export of IkB.

Across many cancer types in adults, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Using proteomic profiling of primary tumor material from patients with high-risk neuroblastoma, as well as gene expression profiling from independent cohorts, we have demonstrated that XPO1 overexpression correlates with poor patient prognosis. Neuroblastoma cell lines are also sensitive to selinexor in the low nanomolar range. Based on these findings and knowledge that bortezomib, a proteasome inhibitor, blocks degradation of XPO1 cargo proteins, we hypothesized that combination treatment with selinexor and bortezomib would synergistically inhibit neuroblastoma cellular proliferation. We observed that selinexor promoted nuclear retention of IkB and that bortezomib augmented the ability of selinexor to induce cell-cycle arrest and cell death by apoptosis. This synergy was abrogated through siRNA knockdown of IkB. The synergistic effect of combining selinexor and bortezomib in vitro provides rationale for further investigation of this combination treatment for patients with high-risk neuroblastoma.