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Hepatocellular adenomas (HCA) in infants are exceedingly rare with only 5 cases reported to the best of our knowledge, all of them preceding the classification of HCA. Here we present an autopsy case of a 9-month-old girl with Burn-McKeown syndrome with an incidental liver nodule in the right lobe measuring 1.5 cm in greatest dimension. The lesion was composed of an unencapsulated proliferation of hepatocytes with multiple unaccompanied arteries without well-formed portal tracts, and an intact reticulin framework without thickened hepatic plates, findings consistent with an HCA. Glutamine synthetase (GS), lipid fatty acid-binding protein (LFABP), c-reactive protein (CRP), serum amyloid-a (SAA), beta-catenin and CD34 immunostains were performed. GS was diffusely and strongly positive in the lesion, CD34 showed heterogenous staining of sinusoids within the lesion without a well-formed rim from the background liver and beta-catenin was negative for nuclear staining. CRP and SAA were considered negative, and LFABP was retained. Molecular testing showed no CTNNB1 variants and found two tier 3 variants involving CHEK2 and PTEN genes. These findings are consistent with an unclassified HCA (U-HCA) per the 2019 WHO Classification of Tumors, representing the youngest patient reported. This raises the possibility that some HCAs are congenital or develop very early in life, remaining undiagnosed until later in life.

Context.— Pediatric soft tissue tumors are one of the areas of pediatric pathology that frequently generate consult requests. Evolving classification systems, ancillary testing methods, new treatment options, research enrollment opportunities, and tissue archival processes create additional complexity in handling these unique specimens. Pathologists are at the heart of this critical decision-making, balancing responsibilities to consider expediency, accessibility, and cost-effectiveness of ancillary testing during pathologic examination and reporting. Objective.— To provide a practical approach to handling pediatric soft tissue tumor specimens, including volume considerations, immunohistochemical staining panel recommendations, genetic and molecular testing approaches, and other processes that impact the quality and efficiency of tumor tissue triage. Data Sources.— The World Health Organization Classification of Soft Tissue and Bone Tumors, 5th edition, other recent literature investigating tissue handling, and the collective clinical experience of the group are used in this manuscript. Conclusions.— Pediatric soft tissue tumors can be difficult to diagnose, and evaluation can be improved by adopting a thoughtful, algorithmic approach to maximize available tissue and minimize time to diagnosis.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

We report a mediastinal neuroblastoma in an octogenarian with paraneoplastic syndrome of inappropriate antidiuretic hormone secretion (SIADH). Neuroblastomas are very rare tumors in adults, with thoracic or mediastinal locations being especially uncommon. These neoplasms have been occasionally associated with the SIADH. Given the rarity of incidence and paucity of diagnostic and outcomes data, the significance of standard neuroblastoma prognostic characteristics is unclear, and no treatment paradigms exist for these patients. Further studies are needed to inform future clinical guidelines.

Context.— The 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists (CAP) tier classification guideline provides a framework to standardize interpretation and reporting of somatic variants. Objective.— To evaluate the adoption and performance of the 2017 guideline among laboratories performing somatic next-generation sequencing (NGS). Design.— A survey was distributed to laboratories participating in NGS CAP proficiency testing for solid tumors (NGSST) and hematologic malignancies (NGSHM). Results.— Worldwide, 64.4% (152 of 236) of NGSST and 66.4% (87 of 131) of NGSHM participants used tier classification systems, of which the 2017 guideline was used by 84.9% (129 of 152) of NGSST and 73.6% (64 of 87) of NGSHM participants. The 2017 guideline was modified by 24.4% (30 of 123) of NGSST and 21.7% (13 of 60) of NGSHM laboratories. Laboratories implementing the 2017 guideline were satisfied or very satisfied (74.2% [89 of 120] NGSST and 69.5% [41 of 59] NGSHM), and the impression of tier classification reproducibility was high (mean of 3.9 [NGSST] and 3.6 [NGSHM] on a 5-point scale). Of nonusers, 35.2% (38 of 108) of NGSST and 39.4% (26 of 66) of NGSHM laboratories were planning implementation. For future guideline revisions, respondents favored including variants to monitor disease (63.9% [78 of 122] NGSST, 80.0% [48 of 60] NGSHM) and germline variants (55.3% [63 of 114] NGSST, 75.0% [45 of 60] NGSHM). Additional subtiers were not favored by academic laboratories compared to nonacademic laboratories (P < .001 NGSST and P = .02 NGSHM). Conclusions.— The 2017 guideline has been implemented by more than 50.0% of CAP laboratories. While most laboratories using the 2017 guideline report satisfaction, thoughtful guideline modifications may further enhance the quality, reproducibility, and clinical utility of the 2017 guideline for tiered somatic variant classification.

Diffuse leptomeningeal glioneuronal tumor (DLGNT) occurs predominantly in children and is typically characterized by diffuse leptomeningeal lesions throughout the neuroaxis with focal segments of parenchymal involvement. Recent reports have identified cases without diffuse leptomeningeal involvement that retain classic glioneuronal features on histology. In this report, we present a case of a 4-year-old boy with a large cystic-solid intramedullary spinal cord lesion that on surgical biopsy revealed a biphasic astrocytic tumor with sparsely distributed eosinophilic granular bodies and Rosenthal fibers. Next-generation sequencing revealed a KIAA1549-BRAF fusion, 1p/19q codeletion, and lack of an IDH1 mutation. Methylation profiling demonstrated a calibrated class score of 0.98 for DLGNT and copy number loss of 1p. Despite the morphologic similarities to pilocytic astrocytoma and the lack of oligodendroglial/neuronal components or leptomeningeal dissemination, the molecular profile was definitive in classifying the tumor as DLGNT. This case highlights the importance of molecular and genetic testing in the characterization of pediatric central nervous system tumors.

Kinase alterations are increasingly recognised as oncogenic drivers in mesenchymal tumours. Infantile fibrosarcoma and the related renal tumour, congenital mesoblastic nephroma, were among the first solid tumours shown to harbour recurrent tyrosine kinase fusions, with the canonical ETV6::NTRK3 fusion identified more than 20 years ago. Although targeted testing has long been used in diagnosis, the advent of more robust sequencing techniques has driven the discovery of kinase alterations in an array of mesenchymal tumours. As our ability to identify these genetic alterations has improved, as has our recognition and understanding of the tumours that harbour these alterations. Specifically, this study will focus upon mesenchymal tumours harbouring NTRK or other kinase alterations, including tumours with an infantile fibrosarcoma-like appearance, spindle cell tumours resembling lipofibromatosis or peripheral nerve sheath tumours and those occurring in adults with a fibrosarcoma-like appearance. As publications describing the histology of these tumours increase so, too, do the variety kinase alterations reported, now including NTRK1/2/3, RET, MET, RAF1, BRAF, ALK, EGFR and ABL1 fusions or alterations. To date, these tumours appear locally aggressive and rarely metastatic, without a clear link between traditional features used in histological grading (e.g. mitotic activity, necrosis) and outcome. However, most of these tumours are amenable to new targeted therapies, making their recognition of both diagnostic and therapeutic import. The goal of this study is to review the clinicopathological features of tumours with NTRK and other tyrosine kinase alterations, discuss the most common differential diagnoses and provide recommendations for molecular confirmation with associated treatment implications.

PURPOSE Neurotrophic tyrosine receptor kinase (NTRK) fusions have been described as oncogenic drivers in a variety of tumors. However, little is known about the overall frequency of NTRK fusion in unselected pediatric tumors. Here, we assessed the frequency, fusion partners, and clinical course in pediatric patients with NTRK fusion–positive tumors. PATIENTS AND METHODS We studied 1,347 consecutive pediatric tumors from 1,217 patients who underwent tumor genomic profiling using custom-designed DNA and RNA next-generation sequencing panels. NTRK fusions identified were orthogonally confirmed. RESULTS AND DISCUSSION NTRK fusions were identified in 29 tumors from 27 patients with a positive yield of 2.22% for all patients and 3.08% for solid tumors. Although NTRK2 fusions were found exclusively in CNS tumors and NTRK1 fusions were highly enriched in papillary thyroid carcinomas, NTRK3 fusions were identified in all tumor categories. The most canonical fusion was ETV6-NTRK3 observed in 10 patients with diverse types of tumors. Several novel NTRK fusions were observed in rare tumor types, including KCTD16-NTRK1 in ganglioglioma and IRF2BP2-NTRK3 in papillary thyroid carcinomas. The detection of an NTRK fusion confirmed the morphologic diagnosis including five cases where the final tumor diagnosis was largely based on the discovery of an NTRK fusion. In one patient, the diagnosis was changed because of the identification of an ETV6-NTRK3 fusion. One patient with infantile fibrosarcoma was treated with larotrectinib and achieved complete pathologic remission. CONCLUSION NTRK fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of tumors than previously recognized. These results highlight the importance of screening for NTRK fusions as part of the tumor genomic profiling for patients with pediatric cancer.

The lymphatic system plays an integral part in fluid homeostasis. Disturbances in lymphatic pathways are congenital, posttraumatic, or posttreatment related, such as after Fontan palliation. Lymphatic pathway evaluation is challenging because of the difficulty in introducing contrast material into the lymphatics. Intranodal, intramesenteric, and intrahepatic dynamic contrast-enhanced MR lymphangiography (DCMRL) offer better visualization of major lymphatic pathways. However, these techniques exclude pathways outside the central conduction system, preventing the visualization of abnormalities and, thus, administration of treatment. The authors describe alternative imaging of an axillary pathway via DCMRL in a patient with a symptomatic chylous effusion not previously assessed with current techniques. Keywords: Lymphatic, MR–Dynamic Contrast Enhanced, Pediatrics, Thorax, Pleura Supplemental material is available for this article. © RSNA, 2022

Ossifying fibromyxoid tumor (OFMT) is a rare soft tissue neoplasm of uncertain differentiation that has the capacity for local recurrence and metastasis. Many OFMTs, including typical, atypical, and malignant tumors, have demonstrated recurrent gene fusions. The fusion partners reported to date share a common core function in that they play either a direct or indirect role in processes influencing histone modification. Herein, we report an OFMT with unusual morphology and non-specific immunoprofile harboring a novel MEAF6-SUZ12 fusion. A 34-year-old male presented with a slowly growing mass in the right antecubital fossa. Excision demonstrated a 6.9 cm partially encapsulated, tan-white, lobulated, and calcified lesion. Microscopic evaluation demonstrated cytologically bland spindle to ovoid cells arranged in a haphazard manner within a fibromyxoid background containing dense collagen, often with sclerotic nodules, and randomly distributed ossification. The tumor cells were diffusely positive for CD34 while essentially negative for S100, desmin, MUC4, SOX10, AE1/3, SMA, and EMA. Next-generation sequencing studies (sarcoma gene fusion next-generation sequencing panel with subsequent Sanger confirmation) performed on formalin-fixed paraffin-embedded tissue detected a fusion product between MEAF6 exon 4 (NM_001270875) and SUZ12 exon 2 (NM_001321207.1). The proposed mechanism of pathogenesis in OFMT, namely epigenetic dysregulation, is reinforced by the fact that both of these partner genes are involved in histone modification.

Introduction: Risk of malignancy for pediatric thyroid nodules classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) is not well defined. Correlations between risk of malignancy and ancillary clinical data remain inconclusive. We report a single institutional experience of fine-needle aspiration (FNA) to improve upon current management paradigm of thyroid nodules. Methods: A retrospective chart review of 575 thyroid nodules was performed of 324 patients who underwent 340 FNAs between 2008 and 2018 at the Children’s Hospital of Philadelphia. Demographics, ultrasound (US) characteristics, FNA cytology, surgical pathology, and ancillary data were reviewed. Results: The rate of malignancy according to TBSRTC was 0.0% for category I, 0.8% for category II, 15.6% for category III, 54.5% for category IV, 100.0% for category V, and 100.0% for category VI. The cumulative Thyroid Imaging Reporting and Data System (TI-RADS) score was significantly correlated with benign and malignant nodules on pathology (p < 2.2e−16). Distribution of TI-RADS for cytologically indeterminate nodules with benign or malignant pathology revealed significant differences for composition (p = 3.20e−8) and echogenic foci (p = 0.005) but not for echogenicity (p = 0.445), shape (p = 0.160), margins (p = 0.220), and size (p = 0.105). Distributions of thyroid-stimulating hormone levels between benign and malignant patients was significant (p = 1.58e−3). Conclusions: Nodules with TI-RADS scores >3 should undergo FNA, irrespective of size; surgical resection is recommended for nodules classified as TBSRTC category IV and V due to high risk of malignancy. US surveillance instead of FNA can be performed for nodules with TI-RADS scores ≤3.

Angiosarcomas are rare, malignant soft tissue tumors in children that arise in a wide range of anatomical locations and have limited targeted therapies available. Here, we report a rare case of a pediatric angiosarcoma (pAS) with Li-Fraumeni syndrome (LFS) expressing a novel

Diffuse leptomeningeal glioneuronal tumor (DLGNT) is characterized by widespread leptomeningeal lesions with focal parenchymal involvement. We present a child with a large intramedullary spinal cord lesion, whose biopsy revealed a biphasic astrocytic tumor consistent with pilocytic astrocytoma. Next-generation sequencing revealed a KIAA1549-BRAF fusion, 1p/19q co-deletion, and no IDH1 mutation. Methylation profiling demonstrated a calibrated class score of 0.98 for DLGNT. Despite morphologic similarities to pilocytic astrocytoma and lack of oligodendroglial/neuronal components or leptomeningeal dissemination, the molecular profile definitively classified the tumor as DLGNT. This case highlights the importance of molecular and genetic testing in diagnosing pediatric central nervous system tumors.

Introduction: The diagnostic utility of molecular profiling for the evaluation of indeterminate pediatric thyroid nodules is unclear. We aimed to assess pediatric cases with indeterminate thyroid fine-needle aspiration (FNA) alongside clinicopathologic features and mutational analysis. Methods: A retrospective review of 126 patients with indeterminate cytology who underwent FNA between January 2010 and December 2021 at the Children’s Hospital of Philadelphia was performed. Indeterminate cases defined by The Bethesda System for Reporting Thyroid Cytopathology (AUS/FLUS or TBSRTC III; FN/SFN or TBSRTC IV; SM or TBSRTC V) were correlated to clinicopathologic and genetic characteristics. Results: Of the 114 surgical cases, 48% were malignant, with the majority of malignant cases diagnosed as follicular variant of papillary thyroid carcinoma (28/55). Risk of malignancy increased with TBSRTC category: 23% for AUS/FLUS, 51% for FN/SFN, and 100% for SM nodules. There were significant differences in surgical approach (p < 0.01), performance of lymph node dissection (p < 0.01), histological diagnosis (p < 0.01), primary tumor focality/laterality (p = 0.04), and lymphatic invasion (p = 0.02) based on TBSRTC classification, with resultant differences in post-surgical risk stratification per American Thyroid Association (ATA) Pediatric Guidelines (p = 0.01). Approximately 89% (49/55) of cases were classified as ATA low risk, and 5 of 6 patients with ATA intermediate- or high-risk disease had SM cytology. Somatic molecular testing was performed in 40% (51/126) of tumors; 77% (27/35) of malignant cases and 38% (6/16) of benign cases harbored driver alteration(s). Of the driver-positive malignant cases, 52% (14/27) were associated with low risk (DICER1, PTEN, RAS, and TSHR mutations), 33% (9/27) were associated with high risk (BRAF mutations and ALK, NTRK, and RET fusions), and 15% (4/27) had unreported risk for invasive disease (APC, BLM, and PPM1D mutations and TG-FGFR1 fusion). Incidence of high-risk drivers increased with TBSRTC category. Approximately 23% (8/35) of patients harboring thyroid malignancy did not have an identifiable driver alteration. Conclusions: Molecular analysis is useful to discriminate benign and malignant thyroid nodules with indeterminate cytology. Patients with driver genetic alteration(s) and indeterminate cytology should consider surgical management secondary to the high incidence (82%; 27/33) of thyroid malignancy in these patients.

The group of CNS mesenchymal (non‐meningothelial) and primary glial/neuronal tumors in association with EWSR1‐non‐ETS rearrangements comprises a growing spectrum of entities, mostly reported in isolation with incomplete molecular profiling. Archival files from three pediatric institutions were queried for unusual cases of pediatric (≤21 years) CNS EWSR1‐rearranged tumors confirmed by at least one molecular technique. Extra‐axial tumors and cases with a diagnosis of Ewing sarcoma (EWSR1‐ETS family fusions) were excluded. Additional studies, including anchored multiplex‐PCR with next‐generation sequencing and DNA methylation profiling, were performed as needed to determine fusion partner status and brain tumor methylation class, respectively. Five cases (median 17 years) were identified (M:F of 3:2). Location was parenchymal (n = 3) and undetermined (n = 2) with topographic distributions including posterior fossa (n = 1), frontal (n = 1), temporal (n = 1), parietal (n = 1) and occipital (n = 1) lobes. Final designation with fusion findings included desmoplastic small round cell tumor (EWSR1‐WT1; n = 1) and tumors of uncertain histogenesis (EWSR1‐CREM, n = 1; EWSR1‐CREB1, n = 1; EWSR1‐PLAGL1, n = 1; and EWSR1‐PATZ1, n = 1). Tumors showed a wide spectrum of morphology and biologic behavior. For EWSR1‐CREM, EWSR1‐PLAGL1 and EWSR1‐PATZ1 tumors, no significant methylation scores were reached in the known brain tumor classes. Available outcome (4/5) was reported as favorable (n = 2) and unfavorable (n = 2) with a median follow‐up of 30 months. In conclusion, we describe five primary EWSR1‐non‐ETS fused CNS tumors exhibiting morphologic and biologic heterogeneity and we highlight the clinical importance of determining specific fusion partners to improve diagnostic accuracy, treatment and monitoring. Larger prospective clinicopathological and molecular studies are needed to determine the prognostic implications of histotypes, anatomical location, fusion partners, breakpoints and methylation profiles in patients with these rare tumors., Pediatric EWSR1‐non‐ETS tumors of the CNS are rare and have not been addressed comprehensively, apart from conventional Ewing sarcoma. Here, we characterize the pathological and molecular features of five such tumors.

Aims The classification of paediatric spindle mesenchymal tumours is evolving, and the spectrum of so-called 'infantile fibrosarcoma' has expanded to include tumours with NTRK, BRAF and MET gene fusions. RET-rearranged paediatric spindle cell neoplasms are an emerging group; there is sparse literature on their clinical, pathological and genetic features, and their nosological place in the canon of soft tissue tumours is uncertain. In this study, we report five RET-rearranged paediatric spindle cell tumours with fusion partners MYH10, KIAA1217 and CLIP2. Methods and results The tumours occurred in the pelvic region, paraspinal region, kidney and subcutaneous tissue of hand and abdomen. The patients' ages ranged from 6 months to 13 years (median 1 year). The tumours were composed of monomorphic spindle cells arranged in a fascicular pattern. Lesional cells had minimally atypical ovoid or tapered nuclei and pale cytoplasm with indistinct borders. Necrosis was not identified. Mitoses numbered three to 12 per 10 high-power field. Cases showed inconsistent and variable expression of S100, CD34 and SMA. Clinical behaviour ranged from small lesions potentially cured by simple resection to large lesions exhibiting metastasis, but responsive to kinase inhibitor therapy. Conclusions Our findings help to define RET-rearranged spindle cell tumours. Although it is likely that these tumours comprise part of the morphological and clinical spectrum of infantile fibrosarcoma (IFS), identification of RET gene alteration is important for its unique therapeutic implications.

Tatton-Brown Rahman syndrome (TBRS) is an overgrowth-intellectual disability syndrome caused by heterozygous variants in DNMT3A. Seventy-eight individuals have been reported with a consistent phenotype of somatic overgrowth, mild to moderate intellectual disability, and similar dysmorphisms. We present six individuals with TBRS, including the youngest individual thus far reported, first individual to be diagnosed with tumor testing and two individuals with variants at the Arg882 domain, bringing the total number of reported cases to 82. Patients reported herein have additional clinical features not previously reported in TBRS. One patient had congenital diaphragmatic hernia. One patient carrying the recurrent p.Arg882His DNMT3A variant, who was previously reported as having a phenotype due to a truncating variant in the CLTC gene, developed a ganglioneuroblastoma at 18 months and T-cell lymphoblastic lymphoma at 6 years of age. Four patients manifested symptoms suggestive of autonomic dysfunction, including central sleep apnea, postural orthostatic hypotension, and episodic vasomotor instability in the extremities. We discuss the molecular and clinical findings in our patients with TBRS in context of existing literature.

The PLEKHH2::ALK fusion is a rarely reported gene fusion identified predominantly in lung adenocarcinomas. Tumors with this fusion have been reported to be of durable response to ALK inhibitors. We herein present the case of a 21-year-old woman with a histomorphologically heterogenous mesenchymal neoplasm of the pelvis, expressing both s100 and CD34, with subsequently identified PLEKHH2::ALK fusion. To our knowledge, only a single mesenchymal neoplasm with this gene fusion has been previously reported. We propose that this tumor represents one with a novel ALK fusion in the emerging family of s100 and CD34 expressing mesenchymal neoplasms with oncogenic kinase alterations akin to NTRK -rearranged mesenchymal neoplasms, rather than inflammatory myofibroblastic tumor. Importantly, this tumor demonstrated a significant response to the ALK inhibitor brigatinib.

Importance The current recommendation for pediatric patients with papillary thyroid cancer (PTC) is a total thyroidectomy. This recommendation applies to all stages of PTC, including papillary thyroid microcarcinoma (≤1 cm, T1a) tumors. Objective To evaluate the characteristics of American Joint Committee on Cancer T1 PTC tumors in a large pediatric population and to identify a subgroup of patients who may benefit from a thyroid lobectomy instead of a total thyroidectomy. Design, Setting, and Participants This retrospective cohort study was conducted from January 1, 2009, to May 31, 2020. The study took place at a tertiary care medical center and included 102 patients who were surgically treated for T1 PTC: 52 with stage T1a (≤1 cm) tumors and 50 with stage T1b (>1 cm but ≤2 cm) tumors. Main Outcomes and Measures Primary outcomes included the presence of bilateral disease and lymph node metastasis. Results A total of 102 patients (mean age, 15.3 years [range, 9.7-18.9 years]; 84 girls [82.4%]) were included in the analysis. Among 52 patients with T1a tumors, 10 (19.2%) had bilateral disease, and 15 (28.8%) had central neck lymph node (N1a) metastasis. Among 50 patients with T1b tumors, 10 (20%) had bilateral and 13 (26%) had N1a disease. Of those with T1a, unilateral multifocality was associated with bilateral disease (odds ratio [OR], 2.1; 95% CI, 1.3-3.4) and N1a disease (OR, 5.1; 95% CI, 1.5-17.6). Both N1a disease (OR, 20.0; 95% CI, 3.5-115.0) and ≥4 positive lymph nodes (OR, 8.6; 95% CI, 1.2-60.9) were associated with bilateral disease. In patients with no pathologic evidence of lymph node metastasis (N0), there was a 95% rate of unilateral PTC. In patients with T1b tumors, unilateral multifocality was also associated with bilateral disease (OR, 1.8; 95% CI, 1.3-2.7). Patients with T1b tumors had an increased risk of lateral (N1b) neck lymph node metastasis when compared with those with T1a tumors (OR, 3.7; 95% CI, 1.0-14.5). Conclusions and Relevance The findings of this cohort study suggest that, in patients with unifocal T1a PTC without clinically evident nodal disease on preoperative ultrasonography, a thyroid lobectomy and central neck dissection may be considered. If there is no evidence of unilateral multifocality or if there are fewer than 4 positive lymph nodes on postoperative pathology, then close observation may be considered. These findings have substantial clinical implications and may result in practice changes regarding the extent of thyroid surgery on low-stage pediatric PTC.

Context.— Neoplastic cellularity assessment has become an essential component of molecular oncology testing; however, there are currently no best practice recommendations or guidelines for this potentially variable step in the testing process. Objective.— To describe the domestic and international practices of neoplastic cellularity assessment and to determine how variations in laboratory practices affect neoplastic cellularity assessment accuracy. Design.— Data were derived from 57 US and international laboratories that participated in the 2019 College of American Pathologists Neoplastic Cellularity Proficiency Testing Survey (NEO-B 2019). NEO-B 2019 included 29 laboratory practice questions and 5 images exhibiting challenging histologic features. Participants assessed the neoplastic cellularity of hematoxylin-eosin–stained digital images, and results were compared to a criterion standard derived from a manual cell count. Results.— The survey responses showed variations in the laboratory practices for the assessment of neoplastic cellularity, including the definition of neoplastic cellularity, assessment methodology, counting practices, and quality assurance practices. In some instances, variation in laboratory practice affected neoplastic cellularity assessment performance. Conclusions.— The results highlight the need for a consensus definition and improved standardization of the assessment of neoplastic cellularity. We put forth an initial set of best practice recommendations to begin the process of standardizing neoplastic cellularity assessment.

BackgroundIn 2014, data from a comprehensive multiplatform analysis of 496 adult papillary thyroid cancer samples reported by The Cancer Genome Atlas project suggested that reclassification of thyroid cancer into molecular subtypes, RAS-like and BRAF-like, better reflects clinical behavior than sole reliance on pathological classification. The aim of this study was to categorize the common oncogenic variants in pediatric differentiated thyroid cancer and investigate if mutation subtype classification correlated with the risk of metastasis and response to initial therapy in pediatric DTC.MethodsSomatic cancer gene panel analysis was completed on DTC from 131 pediatric patients. DTC were categorized into RAS-mutant (H-K-NRAS), BRAF-mutant (BRAF p.V600E) and RET/NTRK fusion (RET, NTRK1 and NTRK3 fusions) to determine differences between subtype classification in regard to pathological data (AJCC TNM) as well as response to therapy 1-year after initial treatment had been completed.ResultsMutation-based subtype categories were significant in most variables, including age at diagnosis, metastatic behavior, and the likelihood of remission at 1-year. Patients with RET/NTRK fusions were significantly more likely to have advanced lymph node and distant metastasis and less likely to achieve remission at one year than patients within RAS- or BRAF-mut subgroups.ConclusionsOur data supports that genetic subtyping of pediatric DTC more accurately reflects clinical behavior than sole reliance on pathological classification with patients with RET/NTRK fusions having worse outcomes than those with BRAF-mutant disease. Future trials should consider inclusion of molecular subtype into risk stratification.

In the past decade, there have been major advances in knowledge related to mesenchymal tumors, and new genetic alterations are being delineated. We report a mesenchymal spindle cell neoplasm harboring a novel gene fusion in an infant. Histopathologically, the neoplasm shared some features with sclerosing perineurioma, but immunohistochemically, EMA was negative, whereas GLUT1, NK1-C3, and BCOR were positive. Next-generation sequencing revealed a PCMTD1-pleomorphic adenoma gene 1 (PLAG1) fusion. PLAG1 contributes to the expression of a variety of genes implicated in regulating cell proliferation, and PCMTD1 has been related to the development of certain carcinomas. Recently, other soft tissue tumors in young children associated with PLAG1 fusion variants have been reported. Perhaps, mesenchymal neoplasms presenting PLAG1 fusions with different genes would confirm a specific group (PLAG mesenchymal tumours or "plagomas") in the near future.

PURPOSE The diagnosis of cancer predisposition in pediatric patients with cancer is vital for treatment decisions, surveillance, and management of at-risk family members. Somatic tumor testing can identify potential underlying constitutional variants that confer increased cancer risk. Here, we report the characteristics of constitutional variants identified through tumor testing. MATERIALS AND METHODS Data were abstracted from medical record review of 1,023 patients who received in-house somatic tumor testing over a 28-month period. Patients were identified for testing using referral criteria developed as a collaboration between genomic diagnostics, pathology, and oncology. Characteristics of patients who underwent constitutional testing, including family history and variant loss of heterozygosity, were tracked. RESULTS From 1,023 patients who underwent somatic tumor sequencing in a 28-month period, 210 variants were identified in 141 patients (13.8%) that were concerning for cancer predisposition syndromes requiring intervention. A total of 73 variants in 41 patients have undergone clinical confirmatory testing thus far. Of these, 26 variants were confirmed to be constitutionally present (35.6%). Among patients tested, 23 (56.1%) of 41 total patients were diagnosed with a cancer predisposition syndrome. CONCLUSION Our data demonstrate that more than one third of variants in tumor somatic sequencing that were concerning for underlying cancer predisposition were constitutionally confirmed. Overall, somatic tumor testing identified potential cancer predisposition syndromes in pediatric patients, and some would not have been identified on the basis of clinical history alone.

The family of juvenile xanthogranuloma family neoplasms (JXG) with ERK-pathway mutations are now classified within the “L” (Langerhans) group, which includes Langerhans cell histiocytosis (LCH) and Erdheim Chester disease (ECD). Although theBRAFV600E mutation constitutes the majority of molecular alterations in ECD and LCH, only three reported JXG neoplasms, all in male pediatric patients with localized central nervous system (CNS) involvement, are known to harbor theBRAFmutation. This retrospective case series seeks to redefine the clinicopathologic spectrum of pediatric CNS-JXG family neoplasms in the post-BRAF era, with a revised diagnostic algorithm to include pediatric ECD. Twenty-two CNS-JXG family lesions were retrieved from consult files with 64% (n = 14) having informativeBRAFV600E mutational testing (molecular and/or VE1 immunohistochemistry). Of these, 71% (n = 10) were pediatric cases (≤18 years) and half (n = 5) harbored theBRAFV600E mutation. As compared to theBRAFwild-type cohort (WT), theBRAFV600E cohort had a similar mean age at diagnosis [BRAF V600E: 7 years (3–12 y), vs. WT: 7.6 years (1–18 y)] but demonstrated a stronger male/female ratio (BRAF V600E: 4 vs WT: 0.67), and had both more multifocal CNS disease ( BRAFV600E: 80% vs WT: 20%) and systemic disease (BRAF V600E: 40% vs WT: none). Radiographic features of CNS-JXG varied but typically included enhancing CNS mass lesion(s) with associated white matter changes in a subset ofBRAFV600E neoplasms. After clinical-radiographic correlation, pediatric ECD was diagnosed in theBRAFV600E cohort. Treatment options varied, including surgical resection, chemotherapy, and targeted therapy with BRAF-inhibitor dabrafenib in one mutated case.BRAFV600E CNS-JXG neoplasms appear associated with male gender and aggressive disease presentation including pediatric ECD. We propose a revised diagnostic algorithm for CNS-JXG that includes an initial morphologic diagnosis with a final integrated diagnosis after clinical-radiographic and molecular correlation, in order to identify cases of pediatric ECD. Future studies with long-term follow-up are required to determine if pediatricBRAFV600E positive CNS-JXG neoplasms are a distinct entity in the L-group histiocytosis category or represent an expanded pediatric spectrum of ECD.

Context.— There has been a rapid expansion of next-generation sequencing (NGS)–based assays for the detection of somatic variants in solid tumors. However, limited data are available regarding the comparative performance of NGS and non-NGS assays using standardized samples across a large number of laboratories. Objective.— To compare the performance of NGS and non-NGS assays using well-characterized proficiency testing samples provided by the College of American Pathologists (CAP) Molecular Oncology Committee. A secondary goal was to compare the use of preanalytic and postanalytic practices. Design.— A total of 17 343 responses were obtained from participants in the BRAF, EGFR, KRAS, and the Multigene Tumor Panel surveys across 84 different proficiency testing samples interrogating 16 variants and 3 wild-type sequences. Performance and preanalytic/postanalytic practices were analyzed by method. Results.— While both NGS and non-NGS achieved an acceptable response rate of greater than 95%, the overall performance of NGS methods was significantly better than that of non-NGS methods for the identification of variants in BRAF (overall 97.8% versus 95.6% acceptable responses, P = .001) and EGFR (overall 98.5% versus 97.3%, P = .01) and was similar for KRAS (overall 98.8% and 97.6%, P = .10). There were specific variant differences, but in all discrepant cases, NGS methods outperformed non-NGS methods. NGS laboratories also more consistently used preanalytic and postanalytic practices suggested by the CAP checklist requirements than non-NGS laboratories. Conclusions.— The overall analytic performance of both methods was excellent. For specific BRAF and EGFR variants, NGS outperformed non-NGS methods and NGS laboratories report superior adherence to suggested laboratory practices.

The spectrum of tumors with NTRK1/2/3 rearrangements has expanded with widespread use of next generation sequencing (NGS) technology. For many years it was known that a majority of infantile fibrosarcomas (IFS), and their counterpart in the kidney, cellular congenital mesoblastic nephroma, contain the recurrent ETV6-NTRK3 fusion. Sequencing RNA transcripts from IFS and their morphologically similar counterparts in older children and adults has shown rearrangements with other 5' partners combined with NTRK1, NTRK2, and NTRK3 can also occur. For those tumors occurring outside of the infant age group, this has resulted in a proposed new diagnostic entity of "NTRK-rearranged spindle cell neoplasm." The clinical behavior of NTRK rearranged soft tissue tumors varies, though most show localized disease with rare metastases. The pathology of NTRK rearranged tumors exists on a spectrum, with overlapping features of classic infantile fibrosarcoma, lipofibromatosis, and malignant peripheral nerve sheath tumor. In this tumor spectrum, clinical and pathologic predictive factors are largely still to be determined, with no clear association between histologic grade and severity of disease. Of critical importance is detection of the NTRK rearrangement in order to guide treatment in patients with unresectable and metastatic disease. While resection is the definitive treatment, these tumors do show response to targeted TRK kinase inhibitors. Multiple detection methods are available, including immunohistochemistry, FISH, and next generation sequencing, which each have their merits and potential pitfalls. We aim to review the clinical characteristics and histomorphology of mesenchymal tumors with NTRK rearrangements as well as discuss molecular detection methods and diagnostic algorithms specific for soft tissue tumors.

BACKGROUND/OBJECTIVES: Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis. DESIGN/METHODS: To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (

CASE Three female children (11-15 years) presented with painful dorsomedial medial cuneiform masses that did not improve after conservative treatment. Findings were consistent with a diagnosis of medial cuneiform apophysis. After surgical resection, no patients had recurrence, although one continued to have pain from a deep peroneal nerve sensory branch that was resected. One patient had an intramass physis identified. CONCLUSION When surgically removing dorsomedial cuneiform masses, surgeons should decompress overlying sensory nerve branches, detach and subsequently repair muscular attachments, and remove the entire stump to prevent regrowth because some of these masses may be apophyses and not exostoses.

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases expressed on the cell membrane that play crucial roles in cellular lineage commitment, differentiation, proliferation, and apoptosis. Deregulated FGFR signaling is observed in a subset of tumors across various histologies, making FGFRs ideal therapeutic targets. We sought to determine the genetic landscape of FGFR-family variations in a cohort of pediatric and young adult patients with solid tumors. The CHOP Comprehensive Solid Tumor Panel was performed on 1,420 patients. The panel covers 238 cancer genes and screens for single nucleotide variants (SNVs), indels, copy number alterations, and 117 fusion gene partners interrogating over 700 exons for known and novel fusions. Identified variants were categorized and reported according to the AMP/ASCO/CAP guidelines. Fifty-six patients (4.1%), including 47 children and 9 young adults, were found to carry at least one FGFR alteration in their tumors. CNS tumors accounted for most of the cases (51 total, 87.9%), with pilomyxoid astrocytoma/pilocytic astrocytoma and dysembryoplastic neuroepithelial tumor the most common (13 and 12 patients, respectively). Non-CNS solid tumors included rhabdomyosarcoma (4 patients), neuroblastoma/ganglioneuroblastoma (2), and follicular thyroid carcinoma (1). FGFR somatic alterations were found in 56 tumors including 41 SNVs and small indels, 6 internal tandem duplications (ITDs), and 15 fusions genes. The most common SNVs observed were hotspot mutations p.K656E and p.N546K of FGFR1. Sequence alterations in FGFR1 contained 35 SNVs and small indels, mostly gain of function mutations located in the kinase domain, and 6 kinase domain ITDs. One SNV was identified in FGFR2 in the immunoglobulin domain. Two SNVs were reported in FGFR3, both of which were in the fibroblast growth factor receptor family domain, and 3 SNVs were identified in FGFR4, all occurring at the p.V550 codon located on the kinase domain. Companion mutations in non-FGFR genes were detected in 27 tumors, predominantly involving RAS signaling pathway genes including NF1 (14 variants), PIK3CA (8), PTPN11 (6) and PIK3R1 (4). Among fusion variants, FGFR1-TACC1 fusions were found in 5 patients, mostly in pediatric patients. One FGFR3-TACC3 fusion was identified in one young adult patient. Seven pediatric patients tested positive for FGFR2 fusions; all with different 3’ partners. The detection of an FGFR alteration defined or changed the histologic diagnosis for 22 patients. Our results reveal that FGFR alterations account for 4.1% (56/1420) of the patients with solid tumors tested in our laboratory. The majority of the FGFR-positive tumors are low-grade CNS tumors. Further, the identification of FGFR alterations can significantly improve the tumor diagnosis and provide genomic evidence for potential targeted treatment with FGFR inhibitors. Citation Format: Jinhua Wu, Jeffrey Schubert, Feng Xu, Ariel Long, Maha Patel, Netta Golenberg, Weixuan Fu, Kajia Cao, Jiani Chen, Elizabeth H. Denenberg, Elizabeth A. Fanning, Rochelle Bagatell, Theodore W. Laetsch, Adam Resnick, Mariarita Santi, Phillip Jay B. Storm, Minjie Luo, Lea F. Surrey, Yiming Zhong, Marilyn M. Li. The spectrum of FGFR mutations in pediatric and young adult solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5268.

Pediatric histiocytic neoplasms are hematopoietic disorders frequently driven by the BRAF-V600E mutation. Here we identified two BRAF gene fusions (novel MTAP-BRAF and MS4A6A-BRAF) in two aggressive histiocytic neoplasms. In contrast to previously described BRAF fusions, MTAP-BRAF and MS4A6A-BRAF do not respond to the paradox breaker RAF inhibitor (RAFi) PLX8394 due to stable fusion dimerization mediated by the N-terminal fusion partners. This highlights a significant and clinically relevant shift from the current dogma that BRAF-fusions respond similarly to BRAF-inhibitors. As an alternative, we show suppression of fusion-driven oncogenic growth with the pan-RAFi LY3009120 and MEK inhibition.

Pediatric histiocytic neoplasms are hematopoietic disorders frequently driven by the BRAF-V600E mutation. Here, we identified two BRAF gene fusions (novel MTAP-BRAF and MS4A6A-BRAF) in two aggressive histiocytic neoplasms. In contrast to previously described BRAF fusions, MTAP-BRAF and MS4A6A-BRAF do not respond to the paradox breaker RAF inhibitor (RAFi) PLX8394 due to stable fusion dimerization mediated by the N-terminal fusion partners. This highlights a significant and clinically relevant shift from the current dogma that BRAF-fusions respond similarly to BRAF-inhibitors. As an alternative, we show suppression of fusion-driven oncogenic growth with the pan-RAFi LY3009120 and MEK inhibition.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia but is approximately 500 times more likely to develop in children with Down syndrome (DS) through transformation of transient abnormal myelopoiesis (TAM). This study investigates the clinical significance of genomic heterogeneity of AMKL in children with and without DS and in children with TAM. Genomic evaluation of nine patients with DS-related TAM or AMKL, and six patients with non-DS AMKL, included conventional cytogenetics and a comprehensive next-generation sequencing panel for single-nucleotide variants/indels and copy-number variants in 118 genes and fusions involving 110 genes. Recurrent gene fusions were found in all patients with non-DS, including two individuals with complex genomes and either a NUP98–KDM5A or a KMT2A–MLLT6 fusion, and the remaining harbored a CBFA2T3–GLIS2 fusion, which arose from both typical and atypical cytogenetic mechanisms. These fusions guided treatment protocols and resulted in a change in diagnosis in two patients. The nine patients with DS had constitutional trisomy 21 and somatic GATA1 mutations, and those with DS-AMKL had two to four additional clinically significant somatic mutations. Comprehensive genomic characterization provides critical information for diagnosis, risk stratification, and treatment decisions for patients with AMKL. Continued genetic and clinical characterization of these rare cancers will aid in improving patient management.

A case of a true dual-genotype IDH-mutant oligoastrocytoma with two different cell types within a single mass in a young woman is presented. Imaging findings of the left frontal infiltrating glioma predicted the two neoplastic components that were identified upon resection. Tissue examination demonstrated areas of tumor with contrasting histologic and molecular features, including specific IDH1, ATRX, TP53, TERT and CIC mutational profiles, consistent with oligodendroglioma and astrocytoma, respectively. The clinical and radiological course over 17 months from first diagnosis included three surgical resections with slow progression of the astrocytic component, and ultimately chemotherapy and radiation treatments were commenced. Reports of the clinical courses for these rare cases of dual-genotype oligoastrocytomas will inform therapy choices, to optimize benefit while minimizing side effects. The steadily increasing number of cases suggests that the neoplasm might be reconsidered as an official entity by the WHO.

Pediatric histiocytic neoplasms are clonal hematopoietic disorders driven by mutations activating the mitogen-activated protein kinase (MAPK) pathway, such as BRAF-V600E. In non-BRAFV600E cases, we investigated alternative MAPK mutations and found two novel BRAF gene fusions. We investigated the distinct responsiveness of novel BRAF fusions to RAFi therapies and explored the mechanistic basis of such differential responses compared to other BRAF fusions. Two histiocytic patient tumors were analyzed using the CHOP Comprehensive Next-Gen Sequencing Solid Tumor Panel and a targeted RNA-seq panel for 106 fusion partner genes. In the two M- and L-type histiocytic neoplasms assessed, we found novel and rare BRAF gene fusions, MTAP-BRAF and MS4A6A-BRAF, respectively. Both BRAF fusions activated the MAPK/ PI3K pathways and showed homo- and hetero-dimerization with BRAF and the respective N-terminal fusion partner. In contrast to common BRAF fusions, MTAP-BRAF and MS4A6A-BRAF did not respond to PLX8394 due to a lack of disruption of active fusion homo- and hetero-dimers, which was in turn due to the untargeted, stable dimerization mediated by the N-terminal fusion partners. Conversely, we observed robust suppression with LY3009120 that bound fusion dimers and kept them in an inactivate confirmation. MEKi were found to successfully suppress fusion driven signaling and oncogenic phenotypes. Our finding that PLX8394 does not disrupt MTAP-BRAF or MS4A6A-BRAF dimerization due to contribution of N-terminal partners defines a novel paradigm for the distinct mechanisms sought by BRAF fusions in response to RAFi therapy. Overall, this study highlights the unique and differential biology hijacked by BRAF fusions in response to RAFi and further warrants detailed mechanistic classification of BRAF fusions based on their responsiveness to targeted agents.

Background Genetic alterations in multiple cell signaling pathways are involved in the molecular pathogenesis of thyroid cancer. Oncogene mutation testing and gene-expression profiling are routinely used for the preoperative risk management of adult thyroid nodules. In this study, we evaluated the potential value of miRNA biomarkers for the classification of pediatric thyroid lesions. Procedure Double-blind case-control study with 113 resected pediatric lesions: 66 malignant and 47 benign. Quantitative and qualitative molecular data generated with a 10-miRNA expression panel (ThyraMIR) and a next-generation sequencing oncogene panel (ThyGeNEXT) were compared with clinicopathological parameters. Results miRNAs were differentially expressed in benign versus malignant tumors with distinct expression patterns in different histopathology categories. The 10-miRNA classifier identified 39 (59%) malignant lesions with 100% specificity. A positive classifier score was associated with lymph node metastasis, extrathyroidal extension and intrathyroidal spread. Genetic alterations associated with increased risk for malignancy were detected in 35 (53%) malignant cases, 20 positive for point mutations in BRAF, HRAS, KRAS, NRAS, PIK3CA, or TERT and 15 positive for gene rearrangements involving ALK, NTRK3, PPARG, or RET. The 10-miRNA classifier correctly identified 11 mutation-negative malignant cases. The performance of the combined molecular test was 70% sensitivity and 96% specificity with an area under the curve of 0.924. Conclusions These data suggest that the regulatory miRNA pathways underlying thyroid tumorigenesis are similar in adults and children. miRNA expression can identify malignant lesions with high specificity, augment the diagnostic yield of mutation testing, and improve the molecular classification of pediatric thyroid nodules.

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.