A Pediatric Primary Cardiac Spindle Cell Neoplasm With a Rare PDGFRA::USP8 Gene Fusion: A Case Report.

We report a case of a primary cardiac spindle cell neoplasm with concerning histological features and a rare PDGFRA::USP8 gene fusion in a 3 year old boy. The patient presented with a large cardiac mass predominantly in the right ventricle, originating from the ventricular septum. The mass was resected with grossly negative margins. Pathology revealed an unclassified spindle cell neoplasm with a PDGFRA::USP8 gene fusion. This gene fusion has only been previously reported twice in the medical literature, one in a pediatric cardiac sarcoma and the other in an abdominal soft tissue tumor in an adult woman. The patient is alive and well with no evidence of recurrence 11 months after excision.

Prognostic and predictive biomarkers in paediatric solid tumours.

Characterisation of histological, immunohistochemical and molecular prognostic and predictive biomarkers has contributed significantly to precision medicine and better outcomes in the management of paediatric solid tumours. Prognostic biomarkers allow predictions to be made regarding a tumour's aggressiveness and clinical course, whereas predictive biomarkers help determine responses to a specific treatment. This review summarises prognostic biomarkers currently used in the more common paediatric solid tumours, with a brief commentary on the most relevant less common predictive biomarkers. MYCN amplification is the most important genetic alteration in neuroblastoma prognosis, and the histological classification devised by Shimada in 1999 is still used in routine diagnosis. Moreover, a new subgrouping of unfavourable histology neuroblastoma enables immunohistochemical characterisation of tumours with markedly different genetic features and prognosis. The predominant histology and commonly observed cytogenetic abnormalities are recognised outcome predictors in Wilms tumour. Evaluation for anaplasia, which is tightly associated with TP53 gene mutations and poor outcomes, is central in both the International Society of Paediatric Oncology and the Children's Oncology Group approaches to disease classification. Characterisation of distinct genotype-phenotype subclasses and critical mutations has expanded overall understanding of hepatoblastoma outcomes. The C1 subclass hepatoblastoma and CTNNB1 mutations are associated with good prognosis. In contrast, the C2 subclass, NFE2L2 mutations, TERT promoter mutations and high expression of oncofetal proteins and stem cell markers are associated with poor outcomes. Risk stratification in sarcomas is highly variable depending on the entity. The prognosis of rhabdomyosarcoma, for example, primarily depends on histological and molecular characteristics. Advances in our understanding of clinically significant biomarkers will translate into more precise diagnoses, improved risk stratification and more effective and less toxic treatment in this challenging group of patients.

The Incidence of Multiple Fusions in a Series of Pediatric Soft Tissue and Bone Tumors.

BACKGROUND: Next generation sequencing (NGS) has increased the detection of fusion genes in cancer. NGS has found multiple fusions in single tumor samples; however, the incidence of this in pediatric soft tissue and bone tumors (PSTBTs) is not well documented. The aim of this study is to catalogue the incidence of multiple fusions in a series of PSTBTs, and apply a modified gene fusion classification system to determine clinical relevance. METHODOLOGY: RNA from 78 bone and soft tissue tumors and 7 external quality assessment samples were sequenced and analyzed using recently-described Metafusion (MF) software and classified using a modification of previously-published schema for fusion classification into 3 tiers: 1, strong clinical significance; 2, potential clinical significance; and 3, unknown clinical significance. RESULTS: One-hundred forty-five fusions were detected in 85 samples. Fifty-five samples (65%) had a single fusion and 30 (35%) had more than 1 fusion. No samples contained more than 1 tier 1 fusion. There were 40 tier 1 (28%), 36 tier 2 (24%), and 69 (48%) tier 3 fusions. CONCLUSIONS: A significant percentage of PSTBTs harbor more than 1 fusion, and by applying a modified fusion classification scheme, the potential clinical relevance of such fusions can be determined.

Germline TP53 mutations undergo copy number gain years prior to tumor diagnosis.

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with germline TP53 pathogenic variants. Here, we perform whole-genome sequence (WGS) analysis of tumors from 22 patients with TP53 germline pathogenic variants. We observe somatic mutations affecting Wnt, PI3K/AKT signaling, epigenetic modifiers and homologous recombination genes as well as mutational signatures associated with prior chemotherapy. We identify near-ubiquitous early loss of heterozygosity of TP53, with gain of the mutant allele. This occurs earlier in these tumors compared to tumors with somatic TP53 mutations, suggesting the timing of this mark may distinguish germline from somatic TP53 mutations. Phylogenetic trees of tumor evolution, reconstructed from bulk and multi-region WGS, reveal that LFS tumors exhibit comparatively limited heterogeneity. Overall, our study delineates early copy number gains of mutant TP53 as a characteristic mutational process in LFS tumorigenesis, likely arising years prior to tumor diagnosis.

The clinical utility of integrative genomics in childhood cancer extends beyond targetable mutations.

We conducted integrative somatic-germline analyses by deeply sequencing 864 cancer-associated genes, complete genomes and transcriptomes for 300 mostly previously treated children and adolescents/young adults with cancer of poor prognosis or with rare tumors enrolled in the SickKids Cancer Sequencing (KiCS) program. Clinically actionable variants were identified in 56% of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants (54% of patients) were of unanticipated timing and type, with over 20% derived from the germline. Corroborating mutational signatures (SBS3/BRCAness) in patients with germline homologous recombination defects demonstrates the potential utility of PARP inhibitors. Mutational burden was significantly elevated in 9% of patients. Sequential sampling identified changes in therapeutically targetable drivers in over one-third of patients, suggesting benefit from rebiopsy for genomic analysis at the time of relapse. Comprehensive cancer genomic profiling is useful at multiple points in the care trajectory for children and adolescents/young adults with cancer, supporting its integration into early clinical management.

Precision cut intestinal slices, a novel model of acute food allergic reactions.

BACKGROUND: Food allergy affects up to 10% of the pediatric population. Despite ongoing efforts, treatment options remain limited. Novel models of food allergy are needed to study response patterns downstream of IgE-crosslinking and evaluate drugs modifying acute events. Here, we report a novel human ex vivo model that displays acute, allergen-specific, IgE-mediated smooth muscle contractions using precision cut intestinal slices (PCIS). METHODS: PCIS were generated using gut tissue samples from children who underwent clinically indicated surgery. Viability and metabolic activity were assessed from 0 to 24 h. Distribution of relevant cell subsets was confirmed using single nucleus RNA sequencing. PCIS were passively sensitized using plasma from peanut allergic donors or peanut-sensitized non-allergic donors, and exposed to various stimuli including serotonin, histamine, FcɛRI-crosslinker, and food allergens. Smooth muscle contractions and mediator release functioned as readouts. A novel program designed to measure contractions was developed to quantify responses. The ability to demonstrate the impact of antihistamines and immunomodulation from peanut oral immunotherapy (OIT) was assessed. RESULTS: PCIS viability was maintained for 24 h. Cellular distribution confirmed the presence of key cell subsets including mast cells. The video analysis tool reliably quantified responses to different stimulatory conditions. Smooth muscle contractions were allergen-specific and reflected the clinical phenotype of the plasma donor. Tryptase measurement confirmed IgE-dependent mast cell-derived mediator release. Antihistamines suppressed histamine-induced contraction and plasma from successful peanut OIT suppressed peanut-specific PCIS contraction. CONCLUSION: PCIS represent a novel human tissue-based model to study acute, IgE-mediated food allergy and pharmaceutical impacts on allergic responses in the gut.

Two congenital cases of pigmented epithelioid melanocytoma with unique clinical and genetic features.

Pigmented epithelioid melanocytomas (PEM) are intermediate-grade melanocytic lesions with frequent lymph node involvement and rare metastases that tend to follow an indolent course with a favorable outcome. We report two unique cases of congenital PEM with PRKCA fusion transcripts: a multifocal PEM with an aggressive incompletely resectable scalp tumor and a solitary palmar PEM with newly reported ITGB5-PRKCA fusion. Through these case reports and a summary of previously reported cases, we outline the spectrum of disease of PEM and highlight the key clinical and histopathologic features associated with PEM with PRKCA fusion transcripts. We also discuss the treatment options and suggest that surgical excision without further adjuvant systemic treatment is reasonable first-line therapy given the favorable prognosis.

Workload Measurement in Subspecialty Placental Pathology in Canada.

BACKGROUND: Workload measurement is important to help determine optimal staffing and workload distribution for pathology laboratories. The Level 4 Equivalent (L4E) System is the most widely used Anatomical Pathology (AP) workload measurement tool in Canada. However, it was initially not developed with subspecialties in mind. METHODS: In 2016, a Pan-Canadian Pediatric-Perinatal Pathology Workload Committee (PCPPPWC) was organized to adapt the L4E System to assess Pediatric-Perinatal Pathology workload. Four working groups were formed. The Placental Pathology Working Group was tasked to develop a scheme for fair valuation of placental specimens signed out by subspecialists in the context of the L4E System. Previous experience, informal time and motion studies, a survey of Canadian Pediatric-Perinatal Pathologists, and interviews of Pathologists' Assistants (PA) informed the development of such scheme. RESULTS: A workload measurement scheme with average L4E workload values for examination and reporting of singleton and multiple gestation placentas was proposed. The proposal was approved by the Canadian Association of Pathologist - Association canadienne des pathologistes Workload and Human Resources Committee for adoption into the L4E System. CONCLUSION: The development of a workload measurement model for placental specimens provides an average and fair valuation of these specimen types, enabling its use for resource planning and workload distribution.

Performance of the McGill Interactive Pediatric OncoGenetic Guidelines for Identifying Cancer Predisposition Syndromes.

IMPORTANCE: Prompt recognition of a child with a cancer predisposition syndrome (CPS) has implications for cancer management, surveillance, genetic counseling, and cascade testing of relatives. Diagnosis of CPS requires practitioner expertise, access to genetic testing, and test result interpretation. This diagnostic process is not accessible in all institutions worldwide, leading to missed CPS diagnoses. Advances in electronic health technology can facilitate CPS risk assessment. OBJECTIVE: To evaluate the diagnostic accuracy of a CPS prediction tool (McGill Interactive Pediatric OncoGenetic Guidelines [MIPOGG]) in identifying children with cancer who have a low or high likelihood of having a CPS. DESIGN, SETTING, AND PARTICIPANTS: In this international, multicenter diagnostic accuracy study, 1071 pediatric (<19 years of age) oncology patients who had a confirmed CPS (12 oncology referral centers) or who underwent germline DNA sequencing through precision medicine programs (6 centers) from January 1, 2000, to July 31, 2020, were studied. EXPOSURES: Exposures were MIPOGG application in patients with cancer and a confirmed CPS (diagnosed through routine clinical care; n = 413) in phase 1 and MIPOGG application in patients with cancer who underwent germline DNA sequencing (n = 658) in phase 2. Study phases did not overlap. Data analysts were blinded to genetic test results. MAIN OUTCOMES AND MEASURES: The performance of MIPOGG in CPS recognition was compared with that of routine clinical care, including identifying a CPS earlier than practitioners. The tool's test characteristics were calculated using next-generation germline DNA sequencing as the comparator. RESULTS: In phase 1, a total of 413 patients with cancer (median age, 3.0 years; range, 0-18 years) and a confirmed CPS were identified. MIPOGG correctly recognized 410 of 412 patients (99.5%) as requiring referral for CPS evaluation at the time of primary cancer diagnosis. Nine patients diagnosed with a CPS by a practitioner after their second malignant tumor were detected by MIPOGG using information available at the time of the first cancer. In phase 2, of 658 children with cancer (median age, 6.6 years; range, 0-18.8 years) who underwent comprehensive germline DNA sequencing, 636 had sufficient information for MIPOGG application. When compared with germline DNA sequencing for CPS detection, the MIPOGG test characteristics for pediatric-onset CPSs were as follows: sensitivity, 90.7%; specificity, 60.5%; positive predictive value, 17.6%; and negative predictive value, 98.6%. Tumor DNA sequencing data confirmed the MIPOGG recommendation for CPS evaluation in 20 of 22 patients with established cancer-CPS associations. CONCLUSIONS AND RELEVANCE: In this diagnostic study, MIPOGG exhibited a favorable accuracy profile for CPS screening and reduced time to CPS recognition. These findings suggest that MIPOGG implementation could standardize and rationalize recommendations for CPS evaluation in children with cancer.

Round Cell Sarcomas: Newcomers and Diagnostic Approaches.

Undifferentiated sarcomas of soft tissue and bone have been defined as tumors with no identifiable morphologic, immunohistochemical, or molecular features indicating tumor cell origin. In young patients, these tumors frequently have a round or spindle cell morphology. Recently described recurrent translocations within this category have led to the recognition of new molecular subtypes of round cell sarcomas, and several of them have a more aggressive clinical course and less chemosensitivity. Because these "newcomers" are diagnosed based on their molecular characteristics, molecular investigation is key in the diagnosis and optimal treatment of these challenging tumors.

Severe Primary Hyperparathyroidism Caused by Parathyroid Carcinoma in a 13-Year-Old Child; Novel Findings From HRpQCT.

Primary hyperparathyroidism is a condition that occurs infrequently in children. Parathyroid carcinoma, as the underlying cause of hyperparathyroidism in this age group, is extraordinarily rare, with only a few cases reported in the literature. We present a 13-year-old boy with musculoskeletal pain who was found to have brown tumors from primary hyperparathyroidism caused by parafibromin-immunodeficient parathyroid carcinoma. Our patient had no clinical, biochemical, or radiographic evidence of pituitary adenomas, pancreatic tumors, thyroid tumors, pheochromocytoma, jaw tumors, renal abnormalities, or testicular lesions. Germline testing for AP2S1, CASR, CDC73/HRPT2, CDKN1B, GNA11, MEN1, PTH1R, RET, and the GCM2 gene showed no pathological variants, and a microarray of CDC73/HRPT2 did not reveal deletion or duplication. He was managed with i.v. fluids, calcitonin, pamidronate, and denosumab prior to surgery to stabilize hypercalcemia. After removal of a single parathyroid tumor, he developed severe hungry bone syndrome and required 3 weeks of continuous i.v. calcium infusion, in addition to oral calcium and activated vitamin D. Histopathological examination identified an angioinvasive parathyroid carcinoma with global loss of parafibromin (protein encoded by CDC73/HRPT2).HRpQCT and DXA studies were obtained prior to surgery and 18-months postsurgery. HRpQCT showed a resolution of osteolytic lesions combined with structural improvement of cortical porosity and an increase in both cortical thickness and density compared with levels prior to treatment. These findings highlight the added value of HRpQCT in primary hyperparathyroidism. In addition to our case, we have provided a review of the published cases of parathyroid cancer in children. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Abnormal hemostasis in children with vascular anomalies, part I: Thrombocytopenias among different vascular anomalies.

Long before the classification of vascular anomalies from the International Society for the Study of Vascular Anomalies (ISSVA) provided a framework to differentiate vascular anomalies, otherwise known as vascular birthmarks, it was recognized that patients with such lesions can present with acute life-threatening hemostatic and/or thrombotic complications, as well as chronic long-standing bleeding or thrombotic issues. Scenarios such as a rapidly growing vascular lesion with severe acute thrombocytopenia, a visceral hemorrhagic lesion, a lesion associated with repetitive and painful superficial thrombosis, and cases of unprovoked or post-procedural fatal pulmonary embolism highlight the wide spectrum of manifestations of abnormal coagulation in patients with vascular anomalies. The separation of vascular anomalies into two distinct groups, vascular tumors and vascular malformations, was followed by the characterization that their respective coagulopathies were due to either a derangement of platelets or to a disequilibrium of the patient's coagulation/fibrinolytic process. This configuration of coagulopathies will be the foundation for this two-chapter review series. In the initial review, coagulopathies where thrombocytopenia is the main feature will be characterized, whereas the second review will focus on vascular malformations that have a coagulation disorder secondary to some degree of coagulation consumption and/or fibrinolytic pathway derangement.

An eHealth decision-support tool to prioritize referral practices for genetic evaluation of patients with Wilms tumor.

Over 10% of children with Wilms tumor (WT) have an underlying cancer predisposition syndrome (CPS). Cognizant of increasing demand for genetic evaluation and limited resources across health care settings, there is an urgent need to rationalize genetic referrals for this population. The McGill Interactive Pediatric OncoGenetic Guidelines study, a Canadian multi-institutional initiative, aims to develop an eHealth tool to assist physicians in identifying children at elevated risk of having a CPS. As part of this project, a decisional algorithm specific to WT consisting of five tumor-specific criteria (age <2 years, bilaterality/multifocality, stromal-predominant histology, nephrogenic rests, and overgrowth features) and universal criteria including features of family history suspicious for CPS and congenital anomalies, was developed. Application of the algorithm generates a binary recommendation-for or against genetic referral for CPS evaluation. To evaluate the algorithm's sensitivity for CPS identification, we retrospectively applied the tool in consecutive pediatric patients (n = 180) with WT, diagnosed and/or treated at The Hospital for Sick Children (1997-2016). Odds ratios were calculated to evaluate the strengths of associations between each criterion and specific CPS subtypes. Application of the algorithm identified 100% of children with WT and a confirmed CPS (n = 27). Age <2 years, bilaterality/multifocality, and congenital anomalies were strongly associated with pathogenic variants in WT1. Presence of >1 overgrowth feature was strongly associated with Beckwith-Wiedemann syndrome. Stromal-predominant histology did not contribute to CPS identification. We recommend the incorporation of the WT algorithm in the routine assessment of children with WT to facilitate prioritization of genetic referrals in a sustainable manner.

Multiplexed Digital Detection of B-Cell Acute Lymphoblastic Leukemia Fusion Transcripts Using the NanoString nCounter System.

Chromosomal rearrangements resulting in fusion transcripts have been reported in precursor B-cell acute lymphoblastic leukemia (B-ALL). The identification of fusion events is crucial in the diagnosis of B-ALL. In this study, we used NanoString nCounter technology to design, validate, and evaluate a multiplex panel for the detection of B-ALL fusion transcripts. Fifty-one B-ALL fusion transcripts reported in children in the literature were included in the design of the NanoString panel. Twenty-six fusion transcripts were validated using 64 positive-control samples and 74 negative-control samples with 100% sensitivity and 99% specificity in comparison to RT-PCR. Our results support a potential role of NanoString's technology as a robust and cost-effective technique that could be used in the detection of fusion transcripts and implemented into the diagnostic algorithm of B-ALL.

Management of pediatric appendiceal carcinoid: a single institution experience from 5000 appendectomies.

AIM OF THE STUDY: Appendiceal carcinoid (neuroendocrine tumor or NET) is a rare neuroendocrine neoplasm often found incidentally following appendectomy for appendicitis. Surgery for appendicitis is currently under scrutiny and children are increasingly managed conservatively with antibiotics alone. Herein, we aimed to review our experience with the management of appendiceal carcinoids at our institution. METHODS: Following ethical approval, we reviewed the charts of all patients who underwent appendectomy for appendicitis at our institution between 2000 and 2018. The pathology registry was consulted to identify children diagnosed with appendiceal carcinoid. Outcome measures included incidence, demographics, and management. MAIN RESULTS: During the study period, 32 children (23 female) had an appendiceal carcinoid confirmed at pathology. Of these, 13 were initially treated with appendectomy (total of 5,059 appendectomies: 0.3% incidence). The other 19 had an appendectomy elsewhere by an adult general surgeon and were referred to our institution for further management. Overall, the mean age at diagnosis was 13 ± 2.7 years and all patient had a preoperative diagnosis of appendicitis, none of suspected carcinoid. Most children (75%) had acute non-perforated appendicitis. The overall mean size of the lesion was 1 ± 0.9 cm, with a > 2 cm lesion in 3 patients. Following diagnosis, 12 children (38%) underwent an ileocolic resection, due to carcinoid size, invasiveness, and margin clearance. CONCLUSIONS: In our cohort, the incidence of appendiceal carcinoid among children with appendicitis is very low. Most carcinoids are small, located at the tip, associated with non-perforated appendicitis, and present in girls. Most were treated with appendectomy alone, with more extensive surgery performed in one third of children.

Management of orbital rhabdomyosarcoma in a child with Li-Fraumeni syndrome.

This case highlights the management of orbital rhabdomyosarcoma in a child with Li Fraumeni syndrome (LFS). Treatment with chemotherapy and eventual orbital exenteration enabled margin-free control of the tumor. Radiation therapy was avoided to reduce the risk of inducing additional malignancy. Reactive orbital hyperostosis was observed postoperatively and was confirmed with surgical biopsy of the orbital roof. In this case, systemic surveillance imaging, which is necessary in patients with LFS, revealed an adrenal cortical carcinoma.

Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology.

BACKGROUND: NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. DESIGN: Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. RESULTS: Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. CONCLUSION: Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

Burkholderia multivorans septicemia in a pediatric liver transplant patient.

"Cepacia syndrome", caused by Burkholderia cepacia complex and often associated with cystic fibrosis, carries a high mortality rate. It is rare for Burkholderia multivorans, a species within the B. cepacia complex, to cause cepacia syndrome even among patients with cystic fibrosis. This is the first reported fatal case of cepacia syndrome caused by B. multivorans occurring in a pediatric liver transplant recipient who does not have cystic fibrosis. We describe the unique characteristics of this pathogen among the non-cystic fibrosis population and the importance of early recognition and treatment.