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In this study, we evaluate the impact of whole genome and transcriptome analysis (WGTA) on predictive molecular profiling and histologic diagnosis in a cohort of advanced malignancies. WGTA was used to generate reports including molecular alterations and site/tissue of origin prediction. Two reviewers analyzed genomic reports, clinical history, and tumor pathology. We used National Comprehensive Cancer Network (NCCN) consensus guidelines, Food and Drug Administration (FDA) approvals, and provincially reimbursed treatments to define genomic biomarkers associated with approved targeted therapeutic options (TTOs). Tumor tissue/site of origin was reassessed for most cases using genomic analysis, including a machine learning algorithm (Supervised Cancer Origin Prediction Using Expression [SCOPE]) trained on The Cancer Genome Atlas data. WGTA was performed on 652 cases, including a range of primary tumor types/tumor sites and 15 malignant tumors of uncertain histogenesis (MTUH). At the time WGTA was performed, alterations associated with an approved TTO were identified in 39 (6%) cases; 3 of these were not identified through routine pathology workup. In seven (1%) cases, the pathology workup either failed, was not performed, or gave a different result from the WGTA. Approved TTOs identified by WGTA increased to 103 (16%) when applying 2021 guidelines. The histopathologic diagnosis was reviewed in 389 cases and agreed with the diagnostic consensus after WGTA in 94% of non-MTUH cases (n= 374). The remainder included situations where the morphologic diagnosis was changed based on WGTA and clinical data (0.5%), or where the WGTA was non-contributory (5%). The 15 MTUH were all diagnosed as specific tumor types by WGTA. Tumor board reviews including WGTA agreed with almost all initial predictive molecular profile and histopathologic diagnoses. WGTA was a powerful tool to assign site/tissue of origin in MTUH. Current efforts focus on improving therapeutic predictive power and decreasing cost to enhance use of WGTA data as a routine clinical test.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell–omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Background: Arthrochalasia type Ehlers-Danlos Syndrome (EDS) is a connective tissue disease characterized by severe generalized joint hypermobility, congenital bilateral hip dislocations, and recur...

Discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs*18) and presented with joint contractures, osteochondromas, and B cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in pro-survival and inflammatory genes. Systematic single-cell-omic analyses revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (with oncogenic signatures - MYC, JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further define detrimental effects a long-term use of calcineurin inhibitors.

Applications for bowel US in children have been well described; however, less focus has been placed on patterns of bowel wall architectural change in specific disease states. This pictorial essay reviews normal bowel wall architecture and covers a variety of inflammatory, infectious, vascular and neoplastic disorders outside the neonatal period as seen on US, with illustrative pathological correlation. A thorough understanding of normal and abnormal bowel wall architecture can enrich sonographic interpretation and provide a valuable adjunct to appropriate clinical investigation.

Third and fourth branchial anomalies are rare, accounting for less than 10% of all branchial anomalies. The piriform fossa sinus tract (PFST) typically presents with left-side suppurative thyroiditis, although it can present earlier in neonates as a non-inflamed cystic neck mass. PFST poses a considerable diagnostic challenge with variable clinical and imaging features, leading to long delays to definitive diagnosis and appropriate management.To analyse the patterns of presentation and imaging findings in children with PFST, with a particular focus on neonatal presentation.This was a retrospective review of the clinical presentation, imaging findings and management in 16 cases of PFST presenting to our tertiary children's hospital between 2003 and 2018. Cases were identified by medical records and picture archiving and communication system (PACS) search using relevant International Classification of Diseases (ICD)-10 coding.Age at presentation ranged from prenatal to 16 years, with a male-to-female ratio of 2:1. All patients presented with neck swelling. Thirteen patients (81%) had suppurative thyroiditis at initial presentation. Two patients had severe thyroiditis/mediastinitis that required intensive care unit admission. Three neonates presented with noninfected, asymptomatic large cystic neck masses; two of these were detected prenatally and misdiagnosed as lymphatic malformations with subsequent spontaneous clinical resolution that later represented with evidence of PFST. The PFST was on the left side in 15/16 (94%) patients. All patients had neck imaging before definitive diagnosis. Imaging studies included radiographs, ultrasound, computed tomography, magnetic resonance imaging and barium esophagram studies. No single modality was diagnostic of PFST in all patients. Seventy-five percent of patients had multimodal imaging before diagnosis. All PFSTs were confirmed by endoscopic visualisation. Management of PFST was by endoscopic cauterisation in 13 patients and open surgery in 2. One patient did not require surgical correction.Our study highlights the complex nature of PFST. The anomaly is uncommon, has variable clinical and imaging features and may have a lengthy, complicated course if not considered at initial presentation. An episode of suppurative thyroiditis in a child should prompt investigation for PFST. We describe atypical presentations with cystic masses in neonates that appear to resolve but represent later as typical clinical features of PFST.

The programmed death-1 (PD-1) pathway of immune evasion is exploited by many malignancies to limit host T-cell-mediated immune responses. Nivolumab is a PD-1-blocking monoclonal antibody that disrupts this pathway and is FDA approved for the treatment of metastatic melanoma, renal cell carcinoma, and squamous non-small cell lung cancer. In this case report, we describe the first published pediatric experience of nivolumab in refractory classic Hodgkin lymphoma. In this patient with primary refractory disease and high disease burden, cytokine release syndrome requiring inotropic support developed following the first infusion of nivolumab. The patient subsequently demonstrated a dramatic clinical response with resolution of fevers, transfusion independence, improvement in functional status, and very good partial response on PET/CT following a single dose. Nivolumab was continued with corticosteroid and antihistamine premedication without further adverse events and clinical benefit was sustained at 11 months after therapy initiation, despite evidence of slow radiographic disease progression.

Background Information is evolving on liver disease in pediatric patients with Fontan physiology. The purpose of this investigation is to evaluate the spectrum of liver disease in a pediatric population of patients with Fontan physiology and evaluate transient elastography ( TE ) as a noninvasive marker of liver disease. Methods and Results We prospectively enrolled all children with Fontan physiology. All patients underwent comprehensive liver evaluation including liver enzymes (alanine aminotransferase, aspartate aminotransferase, gamma‐glutamyl transferase, alkaline phosphatase), aspartate transaminase to platelet ratio index, albumin, bilirubin, international normalized ratio, complete blood cell count, abdominal ultrasound, and TE . Transjugular liver biopsies and hemodynamic measurements were performed in a subset of patients. A total of 76 children (median, 11.7; interquartile range, 8.4–14.8 [56% male]) were evaluated, with 17 having a transjugular liver biopsy (median 14.8 years; interquartile range, 14.3–17.4). All biopsies showed pathological changes. The severity of liver pathology did not correlate with TE . There was a positive correlation between TE and time since Fontan ( R =0.42, P R =0.29, P =0.02), aspartate transaminase ( R =−0.42, P R =−0.42, P TE ( z =−2.2, P =0.03), low platelet count ( z =1.9, P =0.05), low aspartate transaminase ( z =1.9, P =0.02), and low alkaline phosphatase ( z =2.4, P =0.02). Conclusions Liver disease was ubiquitous in our cohort of pediatric patients with Fontan Physiology. Given the correlation between TE and time from Fontan, TE shows potential as a prospective marker of liver pathology. However, individual measurements with TE do not correlate with the severity of pathology. Given the prevalence of liver disease in this population, protective measures of liver health as well as routine liver health surveillance should be implemented with consideration for hepatology consultation and biopsy in the event of abnormal liver biochemical markers or imaging.

Chordomas are neoplasms derived from notochord cells that typically form in the spine or skull base, and affect both adults and children. Poorly differentiated chordomas, associated with particularly poor outcomes, occur predominantly in children and are characterized by expression of the transcription factor brachyury and loss of INI1/SMARCB1. The use of immune checkpoint inhibitors in pediatric cancer is of growing interest, with reported anti-tumour activity in pediatric Hodgkin lymphoma and gliomas with mismatch repair deficiency. However, responses to immune checkpoint inhibitors are infrequent and unpredictable in pediatric solid tumours, and the molecular markers that can distinguish the seemingly indiscriminate response remain to be defined. Understanding which pediatric patient populations may benefit from immune checkpoint inhibitor therapies is essential. Here we describe the molecular profiles of two pediatric poorly differentiated chordomas using whole-genome, transcriptome and whole-genome bisulfite sequencing as part of the Personalized Oncogenomics program at BC Cancer (NCT02155621) that studies the impact of embedding comprehensive genomic profiling into the clinical care of advanced cancer patients. Genomic profiling and multiplex IHC revealed a high level of CD8+ T cells in both chordoma samples, despite low mutation burden. DNA methylation-based clustering revealed similarities with SMARCB1-deficient rhabdoid tumour subtypes that are have been characterized by increased levels of immune cell infiltration. A dominant tumour-associated T cell receptor clone was identified in one patient sample, which was demonstrated to have affinity for reconstituted brachyury-MHC complexes, indicating a potential anti-tumour immune response directed against brachyury-derived antigens. As these collective observations pointed to a potential benefit of immune targeted therapy, the patient was treated with nivolumab and subsequently achieved a partial radiographic response by RANO criteria. These observations provide support for the benefit of immune checkpoint inhibitors in poorly differentiated chordomas with high levels of infiltrating CD8+ T cells. Citation Format: Laura Williamson, Craig Rive, Daniela Di Francesco, Emma Titmuss, Elizabeth Chun, Scott Brown, Katy Milne, Erin Pleasance, Anna F. Lee, Stephen Yip, David Dix, Daniel Renouf, Robert Holt, Brad H. Nelson, Martin Hirst, Steven Jones, Shahrad Rassekh, Rebecca Deyell, Janessa Laskin, Macro A. Marra. Response to nivolumab in a pediatric chordoma with overexpression of brachyury [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 630.

The use of comprehensive personal genomic data to guide cancer treatment decisions has demonstrated success in recent years. However, its application to pediatric cancer remains challenging. Most pediatric cancer genomes have relatively few somatic aberrations and the recognized oncogenic drivers are often not targetable. Here we describe a whole genome and transcriptome analysis approach to elucidate the impact of currently un-targetable disease drivers on pathways and regulatory networks within cancer cells, in an effort to identify aberrations that can be targeted therapeutically. In the pediatric personalized oncogenomics (PedsPOG) project, we have analyzed 81 pediatric and young adult cases representing diverse types of relapsed, refractory or very poor prognosis cancers (17 primary central nervous system (CNS), 5 hematopoietic and lymphoid malignancies, and 59 from a variety of non-CNS solid cancers). Patients' ages ranged from 0.4-20.7 years at enrollment, with a median of 11.5 years. For each patient, genomic sequencing data from tumour and normal samples were analysed to detect germline and somatic variants including simple mutations to more complex alterations such as gene fusions, mutation burden and mutation signatures. Tumour transcriptome data was analyzed to identify aberrant gene expression and calculate immune-related signatures such as presence and composition of T cells in the tumour sample. Combined interpretation of germline and somatic variants was used to understand the underlying tumour biology and provide rationale for treatment options. Using the integrated analysis of genomic and transcriptomic data, actionable targets were identified in 80 out of 81 patients (98%), while therapeutic targets would have been identified in only 32-50% of the patients if individual genomic variants were used separately (such as mutation, fusion, and copy number changes). RNA expression data appears to be highly informative, from which targets were detected in 90% of the patients, albeit at lower levels of evidence. In particular, prediction of outlier T-cell presence based on expression data was identified as a potential biomarker which led to favorable response to immune checkpoint inhibition. Twenty-four cases received PedsPOG-informed therapy and disease response was evaluated by RECIST or RANO criteria. Complete response was observed in four patients, and thirteen patients demonstrated partial response or stable disease. In conclusion, the use of whole genome sequencing and transcriptome sequencing has yielded novel findings and therapeutic options in children and adolescents with poor prognosis cancer. Citation Format: Yaoqing Shen, Melika Bonakdar, Laura Williamson, Erin Pleasance, Karen Mungall, Richard A. Moore, Andrew J. Mungall, Stephen Yip, Anna F. Lee, Christopher Dunham, Janessa Laskin, Marco M. Marra, Steven J. Jones, Shahrad R. Rassekh, Rebecca J. Deyell. Application of integrated analysis of whole genome sequencing and RNA sequencing to personalized therapy decision making in pediatric and young adult cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 260.

Mutations in the T-box transcription factor TBX4 gene have been reported in patients with Ischiocoxopodopatellar syndrome (MIM# 147891) and childhood-onset pulmonary arterial hypertension. Whole exome sequencing of DNA from a 1 day old deceased newborn, with severe diffuse developmental lung disorder exhibiting features of acinar dysplasia, and her unaffected parents identified a de novo TBX4 missense mutation p.E86Q (c.256G>C) in the DNA-binding T-box domain. We propose phenotypic expansion of the TBX4-related clinical disease spectrum to include acinar dysplasia of the lungs. The reported mutation is the first identified genetic variant causative for acinar dysplasia. © 2016 Wiley Periodicals, Inc.

The etiology of deep dyspareunia in endometriosis is unclear. Our objective was to determine whether nerve bundle density in the cul-de-sac/uterosacrals (zone II) is associated with deep dyspareunia in women with endometriosis. We conducted a blinded retrospective immunohistochemistry study (n = 58) at a tertiary referral center (2011-2013). Patients were stringently phenotyped into a study group and 2 control groups. The study group (tender endometriosis, n = 29) consisted of patients with deep dyspareunia, a tender zone II on examination, and an endometriosis lesion in zone II excised at surgery. Control group 1 (nontender endometriosis, n = 17) consisted of patients without deep dyspareunia, a nontender zone II on examination, and an endometriosis lesion in zone II excised at surgery. Control group 2 (tender nonendometriosis, n = 12) consisted of patients with deep dyspareunia, a tender zone II on examination, and a nonendometriosis lesion (eg, normal histology) in zone II excised at surgery. Protein gene product 9.5 (PGP9.5) immunohistochemistry was performed to identify nerve bundles (nerve fibers surrounded by perineurium) in the excised zone II lesion. PGP9.5 nerve bundle density (bundles/high powered field [HPF]) was then scored by a pathologist blinded to the group. We found a significant difference in PGP9.5 nerve bundle density between the 3 groups (analysis of variance, F2,55 = 6.39, P = .003). Mean PGP9.5 nerve bundle density was significantly higher in the study group (1.16 ± 0.56 bundles/HPF [±standard deviation]) compared to control group 1 (0.65 ± 0.36, Tukey test, P = .005) and control group 2 (0.72 ± 0.56, Tukey test, P = .044). This study provides evidence that neurogenesis in the cul-de-sac/uterosacrals may be an etiological factor for deep dyspareunia in endometriosis.

The placenta is unique among surgical pathology specimens in that it reflects the (patho)physiologies of two patients: the mother and the fetus. Placental examination may offer diagnostic, prognostic, and therapeutic information of clinical relevance to both neonates and their mothers. Pathological processes of relevance to future pregnancies may also be identified, facilitating preconception counseling as well as suggesting monitoring and potential intervention in subsequent gestations. In the setting of intrauterine or neonatal demise, the placenta may be the only, and often most useful, source of information regarding a potential cause of death. The following chapter describes the clinical and pathologic features of both common placental pathologies and uncommon lesions with important clinical implications with which the pathologist should be familiar.

STUDY QUESTION Does incisional endometriosis (IE) harbor somatic cancer-driver mutations? SUMMARY ANSWER We found that approximately one-quarter of IE cases harbor somatic-cancer mutations, which commonly affect components of the MAPK/RAS or PI3K-Akt-mTor signaling pathways. WHAT IS KNOWN ALREADY Despite the classification of endometriosis as a benign gynecological disease, it shares key features with cancers such as resistance to apoptosis and stimulation of angiogenesis and is well-established as the precursor of clear cell and endometrioid ovarian carcinomas. Our group has recently shown that deep infiltrating endometriosis (DE), a form of endometriosis that rarely undergoes malignant transformation, harbors recurrent somatic mutations. STUDY DESIGN, SIZE, DURATION In a retrospective study comparing iatrogenically induced and endogenously occurring forms of endometriosis unlikely to progress to cancer, we examined endometriosis specimens from 40 women with IE and 36 women with DE. Specimens were collected between 2004 and 2017 from five hospital sites in either Canada, Germany or the Netherlands. IE and DE cohorts were age-matched and all women presented with histologically typical endometriosis without known history of malignancy. PARTICIPANTS/MATERIALS, SETTING, METHODS Archival tissue specimens containing endometriotic lesions were macrodissected and/or laser-capture microdissected to enrich endometriotic stroma and epithelium and a hypersensitive cancer hotspot sequencing panel was used to assess for presence of somatic mutations. Mutations were subsequently validated using droplet digital PCR. PTEN and ARID1A immunohistochemistry (IHC) were performed as surrogates for somatic events resulting in functional loss of respective proteins. MAIN RESULTS AND THE ROLE OF CHANCE Overall, we detected somatic cancer-driver events in 11 of 40 (27.5%) IE cases and 13 of 36 (36.1%) DE cases, including hotspot mutations in KRAS, ERBB2, PIK3CA and CTNNB1. Heterogeneous PTEN loss occurred at similar rates in IE and DE (7/40 vs 5/36, respectively), whereas ARID1A loss only occurred in a single case of DE. While rates of detectable somatic cancer-driver events between IE and DE are not statistically significant (P > 0.05), KRAS activating mutations were more prevalent in DE. LIMITATIONS, REASONS FOR CAUTION Detection of somatic cancer-driver events were limited to hotspots analyzed in our panel-based sequencing assay and loss of protein expression by IHC from archival tissue. Whole genome or exome sequencing, or epigenetic analysis may uncover additional somatic alterations. Moreover, because of the descriptive nature of this study, the functional roles of identified mutations within the context of endometriosis remain unclear and causality cannot be established. WIDER IMPLICATIONS OF THE FINDINGS The alterations we report may be important in driving the growth and survival of endometriosis in ectopic regions of the body. Given the frequency of mutation in surgically displaced endometrium (IE), examination of similar somatic events in eutopic endometrium, as well as clinically annotated cases of other forms of endometriosis, in particular endometriomas that are most commonly linked to malignancy, is warranted. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by a Canadian Cancer Society Impact Grant [701603, PI Huntsman], Canadian Institutes of Health Research Transitional Open Operating Grant [MOP-142273, PI Yong], the Canadian Institutes of Health Research Foundation Grant [FDN-154290, PI Huntsman], the Canadian Institutes of Health Research Project Grant [PJT-156084, PIs Yong and Anglesio], and the Janet D. Cottrelle Foundation through the BC Cancer Foundation [PI Huntsman]. D.G. Huntsman is a co-founder and shareholder of Contextual Genomics Inc., a for profit company that provides clinical reporting to assist in cancer patient treatment. R. Aguirre-Hernandez, J. Khattra and L.M. Prentice have a patent MOLECULAR QUALITY ASSURANCE METHODS FOR USE IN SEQUENCING pending and are current (or former) employees of Contextual Genomics Inc. The remaining authors have no competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable.

Clinical detection of sequence and structural variants in known cancer genes points to viable treatment options for a minority of children with cancer.1 To increase the number of children who benefit from genomic profiling, gene expression information must be considered alongside mutations.2,3 Although high expression has been used to nominate drug targets for pediatric cancers,4,5 its utility has not been evaluated in a systematic way.6 We describe a child with a rare sarcoma that was profiled with whole-genome and RNA sequencing (RNA-Seq) techniques. Although the tumor did not harbor DNA mutations targetable by available therapies, incorporation of gene expression information derived from RNA-Seq analysis led to a therapy that produced a significant clinical response. We use this case to describe a framework for inclusion of gene expression into the clinical genomic evaluation of pediatric tumors.

Introduction: Childhood interstitial lung disease (chILD) syndrome describes findings of respiratory symptoms and diffuse abnormalities on lung imaging. Immunodeficiency and immune dysregulation are increasingly recognized as potential causes of these clinical findings. Patients with CD40 ligand (CD40L) deficiency typically present with sinopulmonary and opportunistic infections secondary to impairments in both humoral and cellular immunity. chILD syndrome has not previously been reported as a presenting feature of this disease.Objectives: We describe a patient with CD40L deficiency, caused by a novel mutation in CD40LG (c.464 T>A, p.Leu155Gln), who presented in infancy with chILD syndrome and lung biopsy findings of chronic interstitial pneumonitis and patchy pulmonary glycogenosis.Methods: A left lingula wedge lung biopsy was fixed with formalin and stained for analysis. Whole blood samples from the patient and parents were sent for whole exome sequencing. Flow cytometry to assess CD40L expression on ac...

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

Summary Low-grade fibromyxoid sarcoma was first described more than 20 years ago. Subsequently, it was discovered to carry the recurrent chromosomal translocation t(7;16)(q33;p11) encoding a FUS-CREB3L2 fusion oncoprotein. Molecular tests for this pathognomonic gene fusion can confirm the identity of histologic variants (such as hyalinizing spindle cell tumor with giant rosettes) and suggest that some cases of sclerosing epithelioid fibrosarcoma may represent a high-grade version of this entity. We present a case of an ossifying tumor of the perineum that required an open biopsy and fluorescent in situ hybridization testing for FUS and CREB3L2 for diagnosis as a variant of low-grade fibromyxoid sarcoma. Subsequent excision revealed characteristic areas with collagen rosettes as well as foci of heterotopic ossification. Significant ossification, which is well documented in entities such as synovial sarcoma, ossifying fibromyxoid tumor, and extraskeletal osteosarcoma, has not been reported previously in low-grade fibromyxoid sarcoma. This case demonstrates the value of having a distinctive confirmatory molecular pathology test for diagnosis and expands our knowledge of the histologic variants possible in low-grade fibromyxoid sarcoma.

Children with papillary thyroid carcinoma (PTC) may relapse despite response to radioactive iodine (RAI). Two children with multiply relapsed PTC underwent whole-genome and transcriptome sequencing. A TPM3-NTRK1 fusion was identified in one tumor, with outlier NTRK1 expression compared to the TCGA thyroid cancer compendium and to Illumina BodyMap normal thyroid. This patient demonstrated resolution of multiple pulmonary nodules without toxicity on oral TRK inhibitor therapy. A RET fusion was identified in the second tumor, another potentially actionable finding. Identification of oncogenic drivers in recurrent pediatric PTC may facilitate targeted therapy while avoiding repeated RAI.

Costello syndrome (CS) entails a cancer predisposition and is caused by activating HRAS mutations, typically arising de novo in the paternal germline. Hypoglycemia is common in CS neonates. A previously reported individual with the rare HRAS p.Gln22Lys had hyperinsulinemic hypoglycemia. Autopsy showed a discrete pancreatic nodule. The morphologic and immunohistochemistry findings, including loss of p57(Kip2) protein, were identical to a focal lesion of congenital hyperinsulinism, however, no KCNJ11 or ABCC8 mutation was identified and germline derived DNA showed no alternation of the maternal or paternal 11p15 alleles. Here we report paternal uniparental disomy (pUPD) within the lesion, similar to the pUPD11p15.5 in Beckwith-Wiedemann syndrome (BWS). The similar extent of the pUPD suggests a similar mechanism driving hyperinsulinemia in both conditions. After coincidental somatic LOH and pUPD, the growth promoting effects of the paternally derived HRAS mutation, in combination with the increased function of the adjacent paternally expressed IGF2, may together result in clonal expansion. Although this somatic LOH within pancreatic tissue resulted in hyperinsulinism, similar LOH in mesenchymal cells may drive embryonal rhabdomyosarcoma (ERMS). Interestingly, biallelic IGF2 expression has been linked to rhabdomyosarcoma tumorigenesis and pUPD11 occurred in all 8 ERMS samples from CS individuals. Somatic KRAS and HRAS mutations occur with comparable frequency in isolated malignancies. Yet, the malignancy risk in CS is notably higher than in Noonan syndrome with a KRAS mutation. It is conceivable that HRAS co-localization with IGF2 and the combined effect of pUPD 11p15.5 on both genes contributes to the oncogenic potential.

The p53 family member, p73, is essential for the survival of sympathetic neurons during the developmental period of naturally occurring neuronal death. Here, we have asked whether ΔNp73, which is the only p73 isoform expressed in sympathetic neurons, mediates this survival by p53-dependent and/or p53-independent mechanisms. Initially, we used a genetic approach and crossed p53+/-and p73+/-mice. Quantitation of neurons in the sympathetic superior cervical ganglion during the period of naturally occurring cell death revealed that the loss of p53 partially rescued the death of neurons seen in p73-/-animals. Moreover, exogenous expression of ΔNp73 in cultured p53-/-sympathetic neurons rescued these neurons from apoptosis after NGF withdrawal. Biochemical studies asking how ΔNp73 inhibited NGF withdrawal-induced apoptosis in wild-type neurons demonstrated that it prevented the upregulation of the direct p53 targets p21 and Apaf-1 as well as cleavage of caspase-3. It also inhibited events at the mitochondrial apoptotic checkpoint, suppressing the induction of BimEL and the release of mitochondrial cytochromec. Interestingly, ΔNp73 expression also inhibited one very early event in the apoptotic cascade, the activation of c-Jun N-terminal protein kinase (JNK), likely by binding directly to JNK. Finally, we show that neuronal cell size is decreased in p73-/-mice, and that this decrease is not rescued by the lack of p53, suggesting a role for p73 in regulating cell size that does not involve interactions with p53. Thus, ΔNp73 promotes neuronal survival via p53-dependent and -independent mechanisms, and it does so at multiple points, including some of the most proximal events that occur after NGF withdrawal.

Aim: To investigate feasibility and utility of whole genome and RNA sequencing in cancer care. The use of comprehensive personal genomic information to guide cancer treatment decisions has gained momentum in recent years. Here we present the Personalized Oncogenomics (POG) project launched at British Columbia Cancer Agency in 2012. This project uses paired tumor/normal whole genome and transcriptome sequencing to characterize a patient's tumor and inform treatment options within a clinically relevant time frame. In the past 5 years, around 600 patients with metastatic cancers (including 49 pediatric cases) have been sequenced and analyzed, representing over 50 cancer types. The breadth and depth of data in POG enable the detection of multiple types of alterations, from simple mutations, indels, and copy number changes, to more complex alterations such as gene fusion, mutation signatures, and homologous recombination deficiency score. Incorporation of gene expression data through transcriptome sequencing informs on the impact of observed genomic alterations, and provides information regarding specific diagnosis via expression comparison to other cancer subtypes. All genomic and transcriptomic variants are integrated to build a personalized tumor-specific molecular profile, identify actionable items supported by an in-house knowledgebase and publically available molecular oncology, and characterize each individual tumor by intensive pathway analysis and literature search. Such multidimensional data also impose challenges in interpretation and communication. We developed a pipeline to translate complex genomic data into clinically actionable and hypothetical recommendations for the treatment of individual patients. The pipeline produces two reports: a panel-like report that contains known SNVs and fusions with therapeutic relevance from a collection of more than 4000 events, and a second comprehensive and manually curated report to fully characterize the tumor using the whole genome and transcriptome datasets. Genomic data are presented and discussed at a multidisciplinary molecular tumor board consisting of medical oncologists, pathologists, bioinformaticians, geneticists, and biologists. This approach has enabled clinicians to make informed clinical decisions based on the genomic data integrated with other clinical features, as well as to form new treatment-related hypothesis. POG has shown that use of both whole genome and transcriptome sequencing allows identification of therapeutic targets in a significant proportion of patients. Almost 80% cases were found to have one or more actionable alterations (100% in pediatric cases), and almost one-third of these are defined only using RNA data. Based on molecular tumor board discussion, patients are directed to clinical trials, positioned to genomically informed standard-of-care options, or treated with off-label drugs. With demonstrated effectiveness, the integrative approach developed by POG not only provides molecular insight and treatment options into individual tumors, but also provides a rich resource of molecular data with matched clinical information that will aid our understanding of tumor biology and therapy response mechanisms to inform treatment strategies in the future. Citation Format: Yaoqing Shen, Martin R. Jones, Erin Pleasance, Melika Bonakdar, Carolyn Ch'ng, Caralyn Reisle, Laura Williamson, Elisa Majounie, Greg Taylor, Simon Chan, Brandon Pierce, Wei Zhang, Amir Muhammadzadeh, Eric Y. Zhao, Dustin Bleile, Karen Mungall, Nina Thiessen, Eric Chuah, Tina Wong, Richard Corbett, Yussanne Ma, Richard A. Moore, Andrew J. Mungall, Yongjun Zhao, Stephen Yip, Anna F. Lee, Rod Rassekh, Rebecca Deyell, Howard Lim, Daniel Renouf, Robyn Roscoe, Steven J.M Jones, Janessa Laskin, Marco A. Marra. Clinical application of whole genome and transcriptome sequencing in cancer care [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A184.

Costello syndrome is characterized by constitutional mutations in the proto-oncogene HRAS, causing dysmorphic features, multiple cardiac problems, intellectual disability, and an increased risk of neoplasia. We report a male infant with dysmorphic features, born prematurely at 32 weeks, who, during his 3-month life span, had an unusually severe and ultimately fatal manifestation of hypertrophic cardiomyopathy and hyperinsulinemic hypoglycemia. Molecular studies in this patient demonstrated the uncommon Q22K mutation in the HRAS gene, diagnostic of Costello syndrome. The major autopsy findings revealed hypertrophic cardiomyopathy, congenital myopathy, and a 1.4-cm pancreatic nodule that was positive for insulin expression and morphologically identical to a focal lesion of congenital hyperinsulinism. Sequencing of KCNJ11 and ABCC8, the 2 most commonly mutated genes in focal lesion of congenital hyperinsulinism, revealed no mutations. While hyperinsulinism is a recognized feature of RASopathies, a focal proliferation of endocrine cells similar to a focal lesion of hyperinsulinism is a novel pathologic finding in Costello syndrome.

A variety of immunohistochemical (IHC) stains have been proposed to mark either benign or malignant mesothelial proliferations. Loss of the p16 tumor suppressor (CDKN2A), through homozygous deletions of 9p21, is a good marker of mesotheliomas but lacks sensitivity. Recent reports indicate that some mesotheliomas are associated with loss of BRCA-associated protein 1 (BAP1) expression. Here we investigate BAP1 and p16 as potential markers of malignancy and compare test characteristics with previously proposed markers using a well-characterized tissue microarray. BAP1 protein expression was interrogated by IHC. The p16 locus was examined by fluorescence in situ hybridization (FISH) directed toward chromosome 9p21. Loss of BAP1 was identified in 7/26 mesotheliomas and 0/49 benign proliferations. Loss of p16 was identified in 14/27 mesotheliomas and 0/40 benign proliferations, yielding 100% specificity and positive predictive value for each marker. Together, BAP1 IHC and p16 FISH were 58% sensitive for detecting malignancy. Various combinations of p53, EMA, IMP3, and GLUT1 showed reasonably high specificity (96% to 98%) but poor to extremely poor sensitivity. Combined BAP1 IHC/p16 FISH testing is a highly specific method of diagnosing malignant mesotheliomas when the question is whether a mesothelial proliferation is benign or malignant and is particularly useful when tissue invasion by mesothelial cells cannot be demonstrated. However, combined BAP1/p16 FISH testing is not highly sensitive, and negative results do not rule out a mesothelioma. The test characteristics of previously proposed markers EMA, p53, GLUT1, IMP3 suggest that, even in combination, these markers are not useful tools in this clinical setting.

Here, we show that the p53 family member, p73, is necessary for survival and long-term maintenance of CNS neurons, including postnatal cortical neurons. In p73-/- animals, cortical neuron number is normal at birth but decreases significantly by postnatal day 14 (P14)-P16 because of enhanced apoptosis. This decrease continues into adulthood, when p73-/- animals have approximately one-half as many cortical cells as their wild-type littermates. Cortical neurons express the DeltaNp73alpha protein, and overexpression of DeltaNp73 isoforms rescues cortical neurons from diverse apoptotic stimuli. Thus, DeltaNp73 isoforms are survival proteins in cortical neurons, and their deletion causes a gradual loss of cortical neurons in the weeks and months after birth. This decrease in CNS neuron number in p73-/- animals is not limited to the cortex; facial motor neuron number is decreased, and postnatal development of the olfactory bulb is greatly perturbed. These findings, together with our previous work showing that DeltaNp73 is essential for survival of peripheral sympathetic neurons (Pozniak et al., 2000), indicate that p73 isoforms are essential survival proteins in CNS as well as PNS neurons, and that they likely play a role not only during developmental cell death but also in the long-term maintenance of at least some adult neurons.

Survival rates have plateaued at 70% for osteosarcoma. Proteins ectopically produced by malignant tumors may provide insight into new therapeutic targets. Osteosarcomas secreting human chorionic gonadotropin (hCG) have been suggested to have a worse prognosis. We examined the frequency of expression of β-subunit of hCG (β-hCG) in pretreatment osteosarcoma biopsies, and asked if it was associated with various clinical prognostic parameters, and the development of metastases. We subjected 51 pretreatment biopsies of high-grade osteosarcoma, from 51 patients, to β-hCG immunohistochemistry. In 19 of these patients, postchemotherapy metastatic biopsies also were examined for β-hCG expression. Clinical information (patient age, sex, survival status, and serum hCG in females only), and tumor characteristics (site, size, and presence of metastases) were recorded. The β-hCG positive and negative biopsies were separated and compared. Of 49 interpretable pretreatment biopsies, 28 (57%) showed positive cytoplasmic β-hCG expression: 27 with sparse positivity (1% of tumor cells) and 1 with frequent positivity (10% of tumor cells). The patient with frequent β-hCG positivity in her pretreatment biopsy had elevated serum hCG (88.2 mIU/mL) at diagnosis, decreasing to undetectable following chemotherapy and definitive resection. There was no difference in clinical parameters or rate of metastasis between β-hCG positive versus negative groups. Expression of β-hCG may be seen in high-grade osteosarcoma, but frequent β-hCG immunohistochemical expression by tumor cells, associated with clinically elevated serum β-hCG, is rare. Recognition that some nongerm cell tumors may produce β-hCG can prevent confusion with malignancies containing neoplastic syncytiotrophoblast cells, including germ cell and trophoblastic tumors.

Somatic mutations in the Capicua (CIC) gene were first identified in Type I low-grade gliomas (LGGs), which are characterized by 1p/19q co-deletions and IDH mutations. They are found at frequencies of ~50-70% in this glioma subtype, and have since been identified in ~40% of stomach adenocarcinomas (STADs) of the microsatellite instability (MSI) subtype; however, the role of these somatic mutations in malignancy has yet to be established. In Drosophila, CIC functions as a transcriptional repressor whose activity is inhibited upon activation of the mitogen-activated protein kinase (MAPK) signalling pathway. Though mammalian CIC appears to retain these functions, only three of its target genes have been established in human cells: ETV1, ETV4, and ETV5 (ETV1/4/5). To further probe CIC’s transcriptional network, we developed CIC knockout cell lines and performed transcriptomic and proteiomic analyses in these and in control cell lines expressing wild type CIC, identifying a total of 582 differentially expressed genes. We also used RNA-seq data from The Cancer Genome Atlas (TCGA) for Type I LGGs and STADs to perform additional differential expression analyses between CIC-deficient and CIC-expressing samples. Though gene-level overlap was limited between the three contexts, we found that CIC appears to regulate the expression of genes involved in cell-cell adhesion, metabolism, and developmental processes in all three contexts. These results shed light on the pathological role of CIC mutations and may help explain why these have been associated with poorer outcome within Type I LGGs.

Mutation of the ARID1A gene and loss of the corresponding protein BAF250a has recently been described as a frequent event in clear cell and endometrioid carcinomas of the ovary. To determine whether BAF250a loss is common in other malignancies, immunohistochemistry (IHC) for BAF250a was performed on tissue microarrays (TMAs) in more than 3000 cancers, including carcinomas of breast, lung, thyroid, endometrium, kidney, stomach, oral cavity, cervix, pancreas, colon and rectum, as well as endometrial stromal sarcomas, gastrointestinal stromal tumours, sex cord-stromal tumours and four major types of lymphoma (diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, mantle cell lymphoma and follicular lymphoma). We found that BAF250a loss is frequent in endometrial carcinomas but infrequent in other types of malignancies, with loss observed in 29% (29/101) of grade 1 or 2 and 39% (44/113) of grade 3 endometrioid carcinomas of the endometrium, 18% (17/95) of uterine serous carcinomas and 26% (6/23) of uterine clear cell carcinomas. Since endometrial cancers showed BAF250a loss, we stained whole tissue sections for BAF250a expression in nine cases of atypical hyperplasia and 10 cases of atypical endometriosis. Of the nine cases of complex atypical endometrial hyperplasia, all showed BAF250a expression; however, of 10 cases of atypical endometriosis (the putative precursor lesion for ovarian clear cell and endometrioid carcinoma), one case showed loss of staining for BAF250a in the atypical areas, with retention of staining in areas of non-atypical endometriosis. This was the sole case that recurred as an endometrioid carcinoma, indicating that BAF250a loss may be an early event in carcinogenesis. Since BAF250a loss is seen in endometrial carcinomas at a rate similar to that seen in ovarian carcinomas of clear cell and endometrioid type, and is uncommon in other malignancies, we conclude that loss of BAF250a is a particular feature of carcinomas arising from endometrial glandular epithelium.

Small cell osteosarcoma and mesenchymal chondrosarcoma are 2 primary bone tumors with a small round blue cell component, which can mimic the appearance of Ewing sarcoma. Distinguishing these tumors from each other on biopsy material is important clinically, as optimal therapy differs according to the tumor type. However, separating these entities on morphology alone can be challenging. FLI-1 has been described to be a useful marker for Ewing sarcoma, particularly when hematolymphoid markers are negative. In small cell osteosarcoma and mesenchymal chondrosarcoma, the FLI-1 staining pattern has not been adequately characterized. Using a monoclonal FLI-1 antibody, nuclear immunoreactivity in tumor cells was evaluated in 10 small cell osteosarcomas, 10 mesenchymal chondrosarcomas, and 8 Ewing sarcomas, together with a number of other small, round, blue cell tumors. None of the small cell osteosarcomas or mesenchymal chondrosarcomas exhibited FLI-1 staining in the tumor cells, in contrast to the positive nuclear FLI-1 staining in the stromal endothelial cells. In comparison, 6 of the 8 Ewing sarcomas showed moderate-to-strong nuclear FLI-1 staining of the tumor cells in addition to strong staining of the stromal endothelial cell nuclei. With the exception of lymphoblastic lymphomas, FLI-1 positivity was not seen in the other small round blue cell tumors examined. These findings show that, in contrast to Ewing sarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma lack FLI-1 immunoreactivity. FLI-1 is therefore useful in the differential diagnosis of small round blue cell tumors of the bone.

Hereditary diffuse gastric cancer (HDGC), an autosomal dominant cancer susceptibility syndrome, is largely attributable to germline mutations and deletions in the gene encoding E-cadherin, CDH1. Asymptomatic, mutation-positive individuals often choose prophylactic gastrectomy for cancer risk reduction. Examination of the entire mucosa of prophylactic gastrectomy specimens is essential and shows occult gastric cancers in most cases. We hypothesized that primary screening entire cases stained with periodic acid-Schiff (PAS) instead of hematoxylin and eosin (HE) could improve diagnostic accuracy and speed of detecting invasive signet-ring adenocarcinoma. Serial sections from 6 prophylactic gastrectomy specimens with molecularly confirmed CDH1 mutations were stained with PAS and HE, respectively (108 to 164 blocks per case). PAS-stained and HE-stained slides were randomized for each case and examined microscopically for the presence of invasive signet-ring cells. The time to examine each slide was recorded. Our results showed that significantly fewer lesions were missed (ie, the lesion was initially identified on only 1 section, but present on both sections) on PAS-stained slides (6 missed lesions) than on HE-stained slides (23 missed lesions); (P0.05). Furthermore, it took significantly less time to screen a PAS-stained case (3 h 05+/-41 min) than an HE-stained case (4 h 59+/-1 h 2 min) (P0.05). Selected lesions were confirmed as epithelial by pan-keratin-positive immunohistochemistry. Thus, doing PAS staining instead of HE on CDH1 mutation-positive prophylactic gastrectomy specimens may increase the detection rate of adenocarcinoma while reducing screening time.