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Background: Perivascular tumors, which include myopericytoma and myofibroma, are rare benign soft tissue neoplasms composed of perivascular smooth muscle cells. Most demonstrate characteristic morphology and are readily diagnosed. However, a recently identified hypercellular subset shows atypical histologic features and harbor unique SRF gene fusions. These cellular perivascular tumors can mimic other more common sarcomas with myogenic differentiation. Methods: Clinical, radiological, morphological, immunohistochemical, and molecular findings were reviewed. Results: A slow-growing, fluctuant mass was noted within the philtrum at 16 months. Ultrasonography revealed a well-circumscribed cystic hypoechoic lesion. A small (1.0 cm), tan, well-circumscribed soft-tissue mass was excised after continued growth. Histologically, the encapsulated tumor was hypercellular and composed of spindle cells with predominantly-storiform architecture, focal perivascular condensation, dilated branching thin-walled vessels, increased mitoses, and a smooth muscle immunophenotype. An SRF::NCOA2 fusion was identified. Conclusion: We report the first case of an SRF-rearranged cellular myopericytoma in the perioral region in a young child. This case expands the differential diagnosis of perioral soft tissue tumors with myogenic differentiation. We highlight key clinical, pathological, and molecular features. As we illustrate, these rare tumors pose a considerable diagnostic challenge, and risk misdiagnosis as sarcoma, most notably spindle cell rhabdomyosarcoma.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

Figure S1 shows Tier 1-3 genomic events in INI1-negative tumors

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Purpose:Novel targeted therapeutics have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, with simple tumor genomes and infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The cyclin-dependent kinase (CDK)4/6 pathway recently emerged as a dependency in Ewing sarcoma. Given the heightened efficacy of this class with targeted drug combinations in other cancers, as well as the propensity of resistance to emerge with single agents, we aimed to identify genes mediating resistance to CDK4/6 inhibitors and biologically relevant combinations for use with CDK4/6 inhibitors in Ewing.Experimental Design:We performed a genome-scale open reading frame (ORF) screen in 2 Ewing cell lines sensitive to CDK4/6 inhibitors to identify genes conferring resistance. Concurrently, we established resistance to a CDK4/6 inhibitor in a Ewing cell line.Results:The ORF screen revealed IGF1R as a gene whose overexpression promoted drug escape. We also found elevated levels of phospho-IGF1R in our resistant Ewing cell line, supporting the relevance of IGF1R signaling to acquired resistance. In a small-molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6 inhibitor treatment. The combination of CDK4/6 inhibitors and IGF1R inhibitors was synergistic in vitro and active in mouse models. Mechanistically, this combination more profoundly repressed cell cycle and PI3K/mTOR signaling than either single drug perturbation.Conclusions:Taken together, these results suggest that IGF1R inhibitors activation is an escape mechanism to CDK4/6 inhibitors in Ewing sarcoma and that dual targeting of CDK4/6 inhibitors and IGF1R inhibitors provides a candidate synergistic combination for clinical application in this disease.

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PURPOSE Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS ( P = .04). The most frequently altered genes in the primary cohort were TP 53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort ( P = .015) and the validation cohort ( P = .012). CONCLUSION The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Molecular genetic pathology (MGP) is a subspecialty of pathology and medical genetics and genomics. Genomic testing, which is defined as that which generates large data sets and interrogates large segments of the genome in a single assay, is increasingly recognized as essential for optimal patient care through precision medicine. The most common genomic testing technologies in clinical laboratories are next-generation sequencing and microarray. It is essential to train in these methods and to consider the data generated in the context of the diagnosis, medical history, and other clinical findings of individual patients. Accordingly, updating the MGP fellowship curriculum to include genomics is timely, important, and challenging. At the completion of training, an MGP fellow should be capable of independently interpreting and signing out results of a wide range of genomic assays and, given the appropriate context and institutional support, of developing and validating new assays in compliance with applicable regulations. The Genomics Task Force of the MGP Program Directors, a working group of the Association for Molecular Pathology Training and Education Committee, has developed a genomics curriculum framework and recommendations specific to the MGP fellowship. These recommendations are presented for consideration and implementation by MGP fellowship programs with the understanding that MGP programs exist in a diversity of clinical practice environments with a spectrum of available resources.

PURPOSE Multiple FGFR inhibitors are currently in clinical trials enrolling adults with different solid tumors, while very few enroll pediatric patients. We determined the types and frequency of FGFR alterations ( FGFR1-4) in pediatric cancers to inform future clinical trial design. METHODS Tumors with FGFR alterations were identified from two large cohorts of pediatric solid tumors subjected to targeted DNA sequencing: The Dana-Farber/Boston Children's Profile Study (n = 888) and the multi-institution GAIN/iCAT2 (Genomic Assessment Improves Novel Therapy) Study (n = 571). Data from the combined patient population of 1,395 cases (64 patients were enrolled in both studies) were reviewed and cases in which an FGFR alteration was identified by OncoPanel sequencing were further assessed. RESULTS We identified 41 patients with tumors harboring an oncogenic FGFR alteration. Median age at diagnosis was 8 years (range, 6 months-26 years). Diagnoses included 11 rhabdomyosarcomas, nine low-grade gliomas, and 17 other tumor types. Alterations included gain-of-function sequence variants (n = 19), amplifications (n = 10), oncogenic fusions ( FGFR3:: TACC3 [n = 3], FGFR1:: TACC1 [n = 1], FGFR1:: EBF2 [n = 1], FGFR1:: CLIP2 [n = 1], and FGFR2:: CTNNA3 [n = 1]), pathogenic-leaning variants of uncertain significance (n = 4), and amplification in combination with a pathogenic-leaning variant of uncertain significance (n = 1). Two novel FGFR1 fusions in two different patients were identified in this cohort, one of whom showed a response to an FGFR inhibitor. CONCLUSION In summary, activating FGFR alterations were found in approximately 3% (41/1,395) of pediatric solid tumors, identifying a population of children with cancer who may be eligible and good candidates for trials evaluating FGFR-targeted therapy. Importantly, the genomic and clinical data from this study can help inform drug development in accordance with the Research to Accelerate Cures and Equity for Children Act.

Ultrasound screening for thyroid cancer is recommended in familial adenomatous polyposis (FAP). This study investigated the prevalence of thyroid neoplasia in children with FAP.Cross-sectional study of children with FAP at an academic hospital. Clinical and ultrasound data were analyzed for the prevalence of thyroid nodules and cancer.Of 37 children with FAP, 8 (22%) had thyroid nodules and 2 (5%) had thyroid cancer. Nodules (30%) and cancer (9%) were more common among female subjects and rare among male subjects.Thyroid ultrasound screening in adolescence may benefit female subjects with FAP but has limited utility in male subjects.

Pediatric neoplasms have unique demands, including triaging of small biopsies for multiple testing modalities, and a pediatric cancer genome that is notably different from the adult cancer genome. Pediatric cancers are more likely to be driven by gene fusions and typically have a lower tumor mutational burden. Clinically relevant unique molecular targets exist within pediatric cancers, with important implications for diagnosis, prognosis, and treatment selection. Hence, assays and interpretation workflows must be designed thoughtfully to support molecular tumor profiling for children with cancer, including accommodation of small samples, detection of gene fusions, and consideration of potential germline associations.

Background: Electron microscopy (EM), once an important component in diagnosing pediatric diseases, has experienced a decline in its use. To assess the impact of this, pediatric pathology practices were surveyed regarding EM services. Methods: The Society of Pediatric Pathology Practice Committee surveyed 113 society members from 74 hospitals. Settings included 36 academic tertiary, 32 free-standing children’s, and 6 community hospitals. Results: Over 60% maintained in-house EM services and had more than 2 pathologists interpreting EM while reporting a shortage of EM technologists. Freestanding children’s hospitals had the most specimens (100-200 per year) and more diverse specimen types. Hospitals with fewer than 50 yearly specimens often used reference laboratories. Seventeen had terminated all in-house EM services. Challenges included decreasing caseloads due to alternative diagnostic methods, high operating costs, and shortages of EM technologists and EM-proficient pathologists. Kidney, liver, cilia, heart, and muscle biopsies most often required EM. Lung/bronchoalveolar lavage, tumor, skin, gastrointestinal, nerve, platelet, and autopsy samples less commonly needed EM. Conclusions: The survey revealed challenges in maintaining EM services but demonstrated its sustained value in pediatric pathology. Pediatric pathologists may need to address the centralization of services and training to preserve EM diagnostic proficiency among pathologists who perform ultrastructural interpretations.

Background Ewing sarcoma (EWS) is an aggressive sarcoma with no validated molecular biomarkers. We aimed to determine the frequency of STAG2 protein loss by immunohistochemistry (IHC) and whether loss of expression is associated with outcome. Methods We performed a retrospective cohort study of patients with EWS enrolled to Children’s Oncology Group studies. We obtained unstained slides from 235 patients and DNA for sequencing from 75 patients. STAG2 expression was tested for association with clinical features and survival was estimated using Kaplan–Meier methods with log-rank tests. Results In total, 155 cases passed quality control for STAG2 IHC. STAG2 expression in 20/155 cases could not be categorised with the limited available tissue, leaving 135 patients with definitive STAG2 IHC. In localised and metastatic disease, STAG2 was lost in 29/108 and 6/27 cases, respectively. Among patients with IHC and sequencing, 0/17 STAG2 expressing cases had STAG2 mutations, and 2/7 cases with STAG2 loss had STAG2 mutations. Among patients with localised disease, 5-year event-free survival was 54% (95% CI 34–70%) and 75% (95% CI 63–84%) for patients with STAG2 loss vs. expression (P = 0.0034). Conclusion STAG2 loss of expression is identified in a population of patients without identifiable STAG2 mutations and carries a poor prognosis.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

The spectrum of neoplasms associated with DICER1 variants continues to expand, with the recent addition of primary “DICER1-associated central nervous system sarcoma” (DCS). DCS is a high-grade malignancy predominantly affecting pediatric patients. Six pediatric DCS were identified through a combination of clinical diagnostic studies, archival inquiry, and interinstitutional collaboration. Clinical, histologic, immunohistologic, and molecular features were examined. Genomic findings in the 6 DCS were compared with those in 14 additional DICER1-associated tumors sequenced with the same assay. The six patients presented at ages 3–15 years with CNS tumors located in the temporal (n = 2), parietal (n = 1), fronto-parietal (n = 1), and frontal (n = 2) lobes. All underwent surgical resection. Histologic examination demonstrated high-grade malignant spindle cell tumors with pleuropulmonary blastoma-like embryonic “organoid” features and focal rhabdomyoblastic differentiation; immature cartilage was seen in one case. Immunohistochemically, there was patchy desmin and myogenin staining, and patchy loss of H3K27me3, and within eosinophilic cytoplasmic globules, alfa-fetoprotein staining. Biallelic DICER1 variants were identified in all cases, with germline variants in two of five patients tested. DCS demonstrated genomic alterations enriched for Ras pathway activation and TP53 inactivation. Tumor mutational burden was significantly higher in the 6 DCS tumors than in 14 other DICER1-associated tumors examined (mean 12.9 vs. 6.8 mutations/Mb, p = 0.035). Postoperative care included radiation (n = 5) and chemotherapy (n = 3); at the last follow-up, three patients were alive without DCS, and three had died of disease. Our analysis expands the clinical, histologic, immunohistological, and molecular spectrum of DCS, identifying distinctive features that can aid in the diagnosis, multidisciplinary evaluation, and treatment of DCS.

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.

Cranial fasciitis is a benign myofibroproliferative lesion of the scalp and underlying bones typically occurring in the pediatric population. Histologically, it is characterized by loose fascicles of stellate cells in a fibromyxoid background, findings similar to those described in the closely related variant nodular fasciitis. Previously characterized as a reactive process, the identification of USP6 translocations in over 90% of nodular fasciitis cases prompted their reclassification as a clonal neoplastic process. Unlike nodular fasciitis, the molecular underpinnings of cranial fasciitis are less clear. While a subset of cranial fasciitis has been associated with Wnt/β-catenin pathway dysregulation, recent case reports suggest that this entity may also harbor USP6 fusions, a finding we sought to further investigate. We identified fifteen archival cases of cranial fasciitis, five females and ten males ranging in age from 3 months to 9 years (median 11 months), composed of formalin-fixed paraffin-embedded and fresh frozen tissues (11 and 4 cases respectively). Samples were evaluated on an RNA-based targeted sequencing panel targeting genes recurrently rearranged in neoplasia, including USP6. Five of fifteen cases (33%) were positive for USP6 rearrangements predicted to result in the fusion of the entire USP6 coding region to the promoter of the 5′ partner, (three of which were novel): two SERPINH1-USP6 (novel) and one each of COL3A1-USP6 (novel), SPARC-USP6, and MYH9-USP6. These results demonstrate the recurrent nature of USP6 rearrangements in cranial fasciitis, and highlight the success of targeted RNA sequencing in identifying known and novel fusion partners. The identification of USP6 promoter-swapping rearrangements is helpful in understanding the underlying biology of cranial fasciitis, and reinforces its biologic relationship to nodular fasciitis. Targeted RNA sequencing is a helpful tool in diagnosing this pseudosarcomatous lesion.

The diagnostic distinction between atypical Spitz tumor (AST) and malignant melanoma (MM) in pediatric tumors is challenging. Molecular tests are increasingly used to characterize these neoplasms; however, limited studies are available in pediatric patients. This study aimed to provide a genomic comparison of pediatric MM and AST in the context of comprehensive clinical annotation.Pediatric patients diagnosed with MM (n=11) and AST (n=12) were compared to a cohort of 693 adult melanoma patients. DNA next-generation sequencing assessed kinase gene fusions, tumor mutational burden, sequence variants, copy number alterations, structural variants, microsatellite instability, and mutational signatures.Seven AST cases and eight MM cases were successfully sequenced. Kinase gene fusions were identified in both the MM and AST cohorts (NTRK1, ROS1, and MET). MM cases had TERT, BRAF, and CDKN2A alterations, which were not identified in the AST cohort. Tumor mutational burden (TMB) analysis showed pediatric ASTs had an average of 2.82 mutations/Mb, pediatric MM had an average of 5.7 mutations/Mb, and adult MM cases averaged 18.8 mut/Mb. One pediatric MM case had an elevated TMB of 15 mutations/Mb and a UV mutational signature.These data expand our understanding of pediatric malignant melanoma. The differences between the molecular signatures for AST and MM are not statistically significant, and histopathology remains the gold standard for the diagnosis of pediatric AST and MM at this time. With more data, molecular studies may provide additional support for diagnosis and targeted therapeutics.

PURPOSE Given regulatory approval of immune checkpoint inhibitors in patients with mismatch repair–deficient (MMR-D) cancers agnostic to tumor type, it has become important to characterize occurrence of MMR-D and develop cost-effective screening approaches. Using a next-generation sequencing (NGS) panel (OncoPanel), we developed an algorithm to identify MMR-D frequency in tumor samples and applied it in a clinical setting with pathologist review. METHODS To predict MMR-D, we adapted methods described previously for use in NGS panels, which assess patterns of single base-pair insertion or deletion events occurring in homopolymer regions. Tumors assayed with OncoPanel between July 2013 and July 2018 were included. For tumors tested after June 2017, sequencing results were presented to pathologists in real time for clinical MMR determination, in the context of tumor mutation burden, other mutational signatures, and clinical data. RESULTS Of 20,301 tumors sequenced, 2.7% (553) were retrospectively classified as MMR-D by the algorithm. Of 4,404 samples with pathologist sign-out of MMR status, the algorithm classified 147 (3.3%) as MMR-D: in 116 cases, MMR-D was confirmed by a pathologist, five cases were overruled by the pathologist, and 26 were assessed as indeterminate. Overall, the highest frequencies of OncoPanel-inferred MMR-D were in endometrial (21%; 152/723), colorectal (9.7%; 169/1,744), and small bowel (9.3%; 9/97) cancers. When algorithm predictions were compared with historical MMR immunohistochemistry or polymerase chain reaction results in a set of 325 tumors sequenced before initiation of pathologist assessment, the overall sensitivity and specificity of the algorithm were 91.1% and 98.2%, respectively. CONCLUSION We show that targeted, tumor-only NGS can be leveraged to determine MMR signatures across tumor types, suggesting that broader biomarker screening approaches may have clinical value.

PURPOSE Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.

Pediatric tumors can be divided into hematologic malignancies, central nervous system tumors, and extracranial solid tumors of bone, soft tissue, or other organ systems. Molecular alterations that impact diagnosis, prognosis, treatment, and familial cancer risk have been described in many pediatric solid tumors. In addition to providing a concise summary of clinically relevant molecular alterations in extracranial pediatric solid tumors, this review discusses conventional and next-generation sequencing-based molecular techniques, relevant tumor predisposition syndromes, and the increasing integration of molecular data into the practice of diagnostic pathology for children with solid tumors.

Inflammatory myofibroblastic tumor (IMT) is a rare, mesenchymal tumor that has an increased incidence in childhood. Tumors are usually isolated to the chest, abdomen, and retroperitoneum, but metastatic presentations can be seen. Presenting symptoms are nonspecific and include fever, weight loss, pain, shortness of breath, and cough. Approximately 85% of IMTs harbor actionable kinase fusions. The diagnosis can be delayed because of overlapping features with inflammatory disorders, such as elevated inflammatory markers, increased immunoglobin G levels, fever, weight loss, and morphologic similarity with nonmalignant conditions. We present a girl aged 11 years with a TFG-ROS1 fusion–positive tumor of the lung that was initially diagnosed as an immunoglobin G4–related inflammatory pseudotumor. She underwent complete left-sided pneumonectomy and later recurred with widely metastatic disease. We then report the case of a boy aged 9 years with widely metastatic TFG-ROS1 fusion–positive IMT with rapid molecular diagnosis. In both children, there was an excellent response to oral targeted therapy. These cases reveal that rapid molecular testing of inflammatory tumors is not only important for diagnosis but also reveals therapeutic opportunities. Targeted inhibitors produce significant radiologic responses, enabling potentially curative treatment approaches for metastatic ROS1 fusion IMT with previously limited treatment options. Primary care pediatricians and pediatric subspecialists have a crucial role in the early consultation of a pediatric oncology center experienced in molecular diagnostics to facilitate a comprehensive evaluation for children with inflammatory tumors.

Medullary thyroid carcinoma (MTC) is most commonly associated with RET gene mutations. ALK fusions have rarely been described, although not previously in pediatrics and not previously partnered wit...

Introduction. Myofibromas are rare tumors of pericytic lineage, typically affecting children, and are sometimes aggressive. A subset of sporadic and familial myofibromas have activating variants in PDGFRB. The relationship of myofibroma and PDGFRB to the NOTCH pathway has not yet been described. Methods. Ten myofibroma cases were sequenced with a targeted panel of 447 genes, including copy number variation and selected fusions. Immunohistochemical analysis of total NOTCH3 and activated NOTCH3 was assessed for all 10 myofibroma cases, and a series of histologic mimics (n = 20). Results. Alterations identified by next-generation sequencing included PDGFRB sequence variants in 8/10 cases (80%), a NOTCH3 variant in 1/10 cases (10%), and a NOTCH2 variant in 1/10 cases (10%). All 10 cases also showed a pattern of low-amplitude (1.5- to 2-fold) copy number alterations including gains in PDGFRB and NOTCH3. Ten of 10 myofibromas (100%) showed cytoplasmic staining for total NOTCH3 and 9 of 10 cases (90%) showed nuclear staining for activated NOTCH3. Within the control cohort of histologic mimics, 3 of 3 nodular fasciitis cases (100%) were positive for activated and total NOTCH3, and the remaining 17 cases were negative for pan NOTCH3, while 3 of 3 desmoid-type fibromatosis cases (100%) showed patchy weak nuclear staining for activated NOTCH3. Discussion. Our findings suggest a common pathway of PDGFRB/NOTCH3 activation in myofibromas, even in cases that lack PDGFRB sequence variants. These results support the pericytic lineage of myofibroma. Identification of the characteristic genomic alterations or immunohistochemical staining pattern may facilitate a difficult pathologic diagnosis, and support the use of targeted treatments.

Purpose: Kaposiform lymphangiomatosis (KLA) is a rare, frequently aggressive, systemic disorder of the lymphatic vasculature, occurring primarily in children. Even with multimodal treatments, KLA has a poor prognosis and high mortality rate secondary to coagulopathy, effusions and systemic involvement. We hypothesized that, as has recently been found for other vascular anomalies, KLA may be caused by somatic mosaic variants affecting vascular development. Methods: We performed exome sequencing of tumor samples from five individuals with KLA, along with samples from uninvolved control tissue in three of the five. We used digital PCR (dPCR) to validate the exome findings and to screen KLA samples from six other individuals. Results: We identified a somatic activating NRAS variant (c.182A>G, p.Q61R) in lesional tissue from 10/11 individuals, at levels ranging from 1–28%, that was absent from the tested control tissues. Conclusion: The activating NRAS p.Q61R variant is a known ‘hotspot’ variant, frequently identified in several types of human cancer, especially melanoma. KLA, therefore, joins a growing group of vascular malformations and tumors caused by somatic activating variants in the RAS/PI3K/mTOR signalling pathways. This discovery will expand treatment options for these high risk patients as there is potential for use of targeted RAS pathway inhibitors.

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare aggressive neoplasm that occurs predominantly in children. Like mesenchymal hamartoma of the liver (MHL), UESL harbors recurrent rearrangements involving 19q13.3 and 19q13.4, a region of the genome that contains a primate-specific cluster of micro-RNAs. Here, we present a case of a high-grade neoplasm that arose in the left hepatic lobe of a 5-year-old male and gave rise to widespread lymph node, visceral, and soft tissue metastases. The tumor was composed of sheets, tubules, and papillae of epithelioid cells with rhabdoid morphology. INI1 and BRG1 expression were retained. Tumor cells diffusely expressed epithelial markers, including multiple keratins. While the morphologic and immunophenotypic features were suggestive of poorly differentiated carcinoma with rhabdoid features, the tumor was found to harbor the t(11;19)(q13;q13.3) translocation characteristic of UESL, as well as a TP53 mutation. Given the clinical presentation, imaging, clinical course, the tumor was classified as UESL with unusual, carcinoma-like histopathologic features. In the context of an unclassified high-grade hepatic tumor in a young child, molecular or cytogenetic testing for chromosome 19q13 alterations should be considered.

Introduction/Objective Rhabomyosarcomas (RMS) are a group of skeletal muscle tumors that include embryonal, alveolar, pleomorphic, spindle cell/sclerosing subtypes (SC/SRMS). Spindle cell RMS occurs in both adult and pediatric populations, and is associated with either more aggressive or better clinical outcomes respectively. A recurrent hotspot variant in MYOD1, p.L122R (NM_002478.4 c.365G>T), has been described in SC/SRMS. The classification of this diagnosis is evolving, with VGLL2 and NCOA2 fusions defining the diagnosis in young children, and MYOD1 p.L122R defining the diagnosis in older children. The MYOD1 p.L122R variant seems to be associated with more aggressive disease, and may be increasingly used in risk stratification with intensification of treatment. Methods/Case Report A digital droplet PCR (ddPCR) assay was used to detect the MYOD1 p.L122R in DNA samples with RMS. Patients and controls were coded as positive or negative, and tested for association with clinical features and outcome. Results (if a Case Study enter NA) Known-positive cohort of samples was limited by the extreme rarity of this tumor. “Known-positive” status was established by confirmation of the variant with an external clinically-validated assay. The six known positive samples were assessed by ddPCR for the presence of MYOD1 L122R. The L122R variant was detected in all six variants for a sensitivity of 100%. DNA and/or TNA obtained from known wild-type FFPE and frozen material was assessed, for a total of nine unique samples (1 synthetic, 8 patient-derived). All 9 samples were wild- type, with no positive droplets detected, for a specificity of 100%. Conclusion Our MYOD1 c.365G>T, p.L122R variant detection by droplet digital PCR (ddPCR) assay is a robust, reproducible, specific and sensitive method to detect the MYOD1 hotspot mutation.

Alterations in the BCOR gene, including internal tandem duplications (ITDs) of exon 15 have emerged as important oncogenic changes that define several diagnostic entities. In pediatric cancers, BCOR ITDs have recurrently been described in clear cell sarcoma of kidney (CCSK), primitive myxoid mesenchymal tumor of infancy (PMMTI), and central nervous system high-grade neuroepithelial tumor with BCOR ITD in exon 15 (HGNET-BCOR ITDex15). In adults, BCOR ITDs are also reported in endometrial and other sarcomas. The utility of multiplex targeted RNA sequencing for the identification of BCOR ITD in pediatric cancers was investigated. All available archival cases of CCSK, PMMTI, and HGNET-BCOR ITDex15 were collected. Each case underwent anchored multiplex PCR library preparation with a custom-designed panel, with BCOR targeted for both fusions and ITDs. BCOR ITD was detected in all cases across three histologic subtypes using the RNA panel, with no other fusions identified in any of the cases. All BCOR ITDs occurred in the final exon, within 16 codons from the stop sequence. Multiplex targeted RNA sequencing from formalin-fixed, paraffin-embedded tissue is successful at identifying BCOR internal tandem duplications. This analysis supports the use of anchored multiplex PCR targeted RNA next-generation sequencing panels for identification of BCOR ITDs in pediatric tumors. The use of post-analytic algorithms to improve the detection of BCOR ITD using DNA panels was also explored.

Pediatric pan-cancer genome analyses do not capture the full range of diagnoses encountered in clinical practice. To inform basket trial design and real-world precision oncology practice, we classified diagnoses and assessed the landscape of mutations, including trial-matching, in an unselected cohort of pediatric solid tumors. Since 2013 all Dana-Farber/Boston Children’s patients have been offered participation in the Profile study. Participant tumor samples were sequenced with DFCI-OncoPanel, a targeted panel test sequencing exons of up to 447 cancer genes for single nucleotide variants, insertions and deletions and copy number alterations, and introns and exons of up to 60 genes for rearrangements. Patient diagnosis was classified according to ICD-O, version 3.2. Genomic alterations were analyzed for matching to the actionable mutation lists of precision oncology basket trials (NCI-COG Pediatric MATCH, NCI-MATCH, and the ASCO TAPUR Study v.3). Data will be shared with the Childhood Cancer Data Initiative. There were 888 pediatric patients with sequencing enrolled in Profile between January 2013 and March 2019; 512 (58%) with solid tumors and 376 (42%) with CNS tumors. Fifty-five percent (491/888) of patients had one of ten common pediatric cancer diagnoses: neuroblastoma (n=80), low-grade glioma (n=72), Wilms tumor (n=57), medulloblastoma (n=55), pilocytic astrocytoma (n=47), rhabdomyosarcoma (n=44), osteosarcoma (n=42), ependymoma (n=39), Ewing sarcoma (n=28) and glioblastoma (n=27). The remaining 45% (397/888) had one of 85 distinct rare malignancies with less than 25 cases per diagnosis. Most (80/85) of these rare diagnoses are not represented in prior pediatric pan-cancer sequencing studies. Recurrent (>5%) pathogenic alterations were, in common and rare diagnoses, TP53 mutations(m) and deletions(del) and BRAFm and rearrangements(r), in common diagnoses, MYC/MYCN amplification (amp) and EWSR1r and, in rare diagnoses, CTNNB1m, CDKN2A/Bdel and NF1m/del. We found that 31% (n=271/888) of patients had at least 1 variant matching a basket trial treatment arm. Genes with matching alterations include BRAF (10%), NF1 (4%), PI3KCA (3%), NRAS (2%), BRCA2 (2%), ALK (1%), and FGFR1 (1%). Sequencing of pediatric malignancies is increasing. This study highlights opportunities to use the resulting genomic data to inform genome-selected clinical trial design and uncover drivers in pediatric cancers. The proportion of cases in this cohort with genomic alterations meeting eligibility for basket trials is equivalent to that seen in the pediatric MATCH screening study. Due to the low prevalence of the diagnoses in the long tail of cancer types in this study, defining the genomic landscape of ultra-rare cancers will require data sharing. Classifying pediatric cancer diagnoses using the ICD-O standard ontology system is feasible and will facilitate data sharing. Citation Format: Suzanne J. Forrest, Hersh Gupta, Abigail Ward, Yvonne Li, Duong Doan, Alyaa Al-Ibraheemi, Sanda Alexandrescu, Pratiti Bandopadhayay, Suzanne Shusterman, Elizabeth A. Mullen, Natalie Collins, Susan N. Chi, Karen D. Wright, Priti Kumari, Tali Mazor, Keith L. Ligon, Priyanka Shivdasani, Phani Davineni, Monica Manam, Richard L. Schilsky, Suanna S. Bruinooge, Jaime M. Guidry Auvil, Ethan Cerami, Barrett J. Rollins, Matthew L. Meyerson, Neal I. Lindeman, Laura MacConaill, Bruce E. Johnson, Andrew D. Cherniack, Alanna J. Church, Katherine A. Janeway. Sequencing of 888 pediatric solid tumors informs precision oncology trial design and data sharing initiatives in pediatric cancer [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 3890.

Oncogenic FGFR alterations in the form of activating mutations, amplifications, and fusions are detected in a variety of solid tumors, including those in children. Major advances in FGFR-targeted drug development have led to investigation of multiple generations of FGFR inhibitors in adult clinical trials. In contrast, there are few clinical trials of FGFR inhibitors enrolling children. There is a need to identify and characterize molecular targets including oncogenic FGFR alterations in pediatric cancers so that drug development can occur in accordance with the RACE (Research to Accelerate Cures and Equity) for Children Act. The goal of this study was to determine the frequency and types of FGFR alterations in pediatric cancers to inform future clinical trial design. Tumors with FGFR alterations were identified from two large cohorts of pediatric solid tumors subjected to targeted DNA sequencing: The Dana-Farber/Boston Children’s Profile Study (n=888) and the multi-institution GAIN/iCAT2 (Genomic Assessment Improves Novel Therapy) Study (n=571). Data from the combined patient population of 1,395 cases (64 patients were enrolled in both studies) were reviewed and cases in which an FGFR alteration was identified by OncoPanel sequencing were further assessed. We identified 42 patients with tumors harboring an oncogenic or possibly oncogenic FGFR alteration (FGFR1 in 22; FGFR2 in 4; FGFR3 in 5; FGR4 in 11). Median age at diagnosis was 8 years (range 6 months-26 years old). Diagnoses included 11 rhabdomyosarcomas, 8 low-grade gliomas, 3 high-grade gliomas, and 16 other tumor types. We identified 20 patients with a gain-of-function (GoF) mutation, 10 patients with amplifications (copy number ≥7), 7 patients with fusions, 4 patients with a variant of uncertain significance (VUS) assessed and determined to be possibly oncogenic, and 1 patient with an amplification and VUS. Four of the fusion cases had oncogenic FGFR3-TACC3 (n=3) and FGFR1-TACC1 (n=1) fusions previously reported in the literature. Three cases had novel fusions (FGFR1-EBF2, FGFR1-CLIP2, FGFR2-CTNNA3) found in a spindle cell sarcoma and two low-grade gliomas. Tumors with an FGFR missense mutation were primarily those with oncogenic hotspot mutations in FGFR1 [N546K (n= 5); K656E (n=4)] and FGFR4 [N535K/D (n=2); V550L/E (n=5)]. In summary, this study identified oncogenic FGFR alterations in 42 of 1395 patients (3%) with solid and brain tumors sequenced through the Profile and GAIN/iCAT2 studies who would potentially be eligible for clinical trials evaluating FGFR-targeted therapy. Citation Format: Lorena Lazo De La Vega, Alanna J. Church, Hannah Comeau, Wenjun Kang, AeRang Kim, Navin R. Pinto, Margaret E. Macy, Luke D. Maese, Steven G. DuBois, Bruce E. Johnson, Katherine A. Janeway, Suzanne J. Forrest. Rare FGFR oncogenic alterations in sequenced pediatric solid and brain tumors suggest FGFR is a relevant molecular target in childhood cancer [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 3893.

Cancer of unknown primary (CUP) is a common diagnosis in adult oncology, but is extremely rare in pediatrics, with few published reports and none in the era of molecular profiling. Here, we describe a 13-year old boy with metastatic adenocarcinoma of unknown primary that was refractory to conventional chemotherapy. Molecular profiling revealed an activating ERBB2 D769Y variant. He subsequently achieved disease control on a lapatinib-containing regimen, and upon progression, he transiently responded to neratinib prior to dying from disease. Our case reports a rare example of pediatric CUP and highlights the utility of molecular profiling in these cases.