Publications by authors named "Mary Shago"

61 Publications

Practice patterns of prenatal and perinatal testing in Canadian cytogenetics laboratories.

Prenat Diagn 2021 Apr 21. Epub 2021 Apr 21.

Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Canada.

Objective: To survey patterns of practice in Canadian cytogenetics laboratories and evaluate whether newer technologies have influenced testing algorithms for the detection of common aneuploidies and other genomic imbalances in the prenatal and perinatal settings.

Methods: Cytogenetics laboratories across Canada were invited to participate in two patterns-of-practice surveys: one in 2016 and one in 2019. They were asked to identify the prenatal and perinatal specimen types tested at their facility and which testing methods were used for initial testing and for follow-up.

Results: All clinical laboratories performing prenatal testing offer rapid aneuploidy detection (RAD). Most laboratories also offer microarray analysis. A positive result is either followed up by karyotyping or no further testing is performed. For prenatal samples, a negative result may be followed up by microarray or karyotyping and is dependent on the reason for referral. For perinatal samples, availability of microarray to follow up a negative result is increasing.

Conclusions: Since 2016, the availability of RAD as a first-line test in Canadian cytogenetics laboratories remains consistent, while microarray has become the preferred follow-up testing method over traditional karyotyping following a normal RAD result. Despite a universal healthcare system, disparities in prenatal and perinatal cytogenetic testing algorithms are apparent.
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http://dx.doi.org/10.1002/pd.5951DOI Listing
April 2021

Pediatric fibromyxoid soft tissue tumor with PLAG1 fusion: A novel entity?

Genes Chromosomes Cancer 2021 Apr 30;60(4):263-271. Epub 2020 Dec 30.

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.

The classification of undifferentiated soft tissue tumors continues to evolve with the expanded application of molecular analysis in clinical practice. We report three cases of a unique soft tissue tumor in young children (5 months to 2 years old) displaying a purely fibromyxoid histology, with positive staining for desmin and CD34. In two cases, RNA sequencing detected a YWHAZ-PLAG1 gene fusion, while in the third case, a previously unreported EEF1A1-PLAG1 fusion was identified. PLAG1 fusions have been reported in several pathologic entities including pleomorphic adenoma, myoepithelial tumors of skin and soft tissue, and lipoblastoma, the latter occurring preferentially in young children. In these tumors, expression of a full length PLAG1 protein comes under the control of the constitutively active promoter of the partner gene in the fusion, and the current cases conform to that model. Overexpression of PLAG1 was confirmed by diffusely positive immunostaining for PLAG1 in all three cases. Our findings raise the possibility of a novel fibromyxoid neoplasm in childhood associated with these rare PLAG1 fusion variants. The only other report of a PLAG1-YWHAZ fusion occurred in a pediatric tumor diagnosed as a "fibroblastic lipoblastoma." This finding raises the possibility of a relationship with our three cases, even though our cases lacked any fat component. Further studies with regard to a shared pathogenesis are required.
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http://dx.doi.org/10.1002/gcc.22926DOI Listing
April 2021

First report of t(5;11) KMT2A-MAML1 fusion in de novo infant acute lymphoblastic leukemia.

Cancer Genet 2020 10 22;248-249:31-33. Epub 2020 Sep 22.

Division of Pediatrics Hematology/Oncology, The Hospital for Sick Children; Department of Pediatrics, University of Toronto, Toronto, Canada.

Infant acute lymphoblastic leukemia (ALL) comprises 2.5%-5% of pediatric ALL with inferior survival compared to older children. A majority of infants (80%) with ALL harbor KMT2A gene rearrangement, which portends a poor prognosis. Approximately 94 different partner genes have been identified to date. The common rearrangements include t(4;11)(q21;q23)KMT2A-AFF1,t(11;19) (q23;p13.3)KMT2A-MLLT1 and t(9;11)(p22;q23)KMT2A-MLLT3. We report a novel translocation t(5;11)(q35;q23)KMT2A-MAML1 in newly diagnosed infant precursor B-ALL. Long-term follow-up and a larger number of patients are needed to better understand its prognostic significance.
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http://dx.doi.org/10.1016/j.cancergen.2020.09.004DOI Listing
October 2020

Evidence-based review of genomic aberrations in B-lymphoblastic leukemia/lymphoma: Report from the cancer genomics consortium working group for lymphoblastic leukemia.

Cancer Genet 2020 05 21;243:52-72. Epub 2020 Mar 21.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States.

Clinical management and risk stratification of B-lymphoblastic leukemia/ lymphoma (B-ALL/LBL) depend largely on identification of chromosomal abnormalities obtained using conventional cytogenetics and Fluorescence In Situ Hybridization (FISH) testing. In the last few decades, testing algorithms have been implemented to support an optimal risk-oriented therapy, leading to a large improvement in overall survival. In addition, large scale genomic studies have identified multiple aberrations of prognostic significance that are not routinely tested by existing modalities. However, as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) technologies are increasingly used in clinical management of hematologic malignancies, these abnormalities may be more readily detected. In this article, we have compiled a comprehensive, evidence-based review of the current B-ALL literature, focusing on known and published subtypes described to date. More specifically, we describe the role of various testing modalities in the diagnosis, prognosis, and therapeutic relevance. In addition, we propose a testing algorithm aimed at assisting laboratories in the most effective detection of the underlying genomic abnormalities.
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http://dx.doi.org/10.1016/j.cancergen.2020.03.001DOI Listing
May 2020

Integrated Molecular and Clinical Analysis of 1,000 Pediatric Low-Grade Gliomas.

Cancer Cell 2020 04;37(4):569-583.e5

Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Department of Neurosurgery, The Hospital for Sick Children, Toronto ON, Canada.

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.
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http://dx.doi.org/10.1016/j.ccell.2020.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169997PMC
April 2020

Alterations in ALK/ROS1/NTRK/MET drive a group of infantile hemispheric gliomas.

Nat Commun 2019 09 25;10(1):4343. Epub 2019 Sep 25.

Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic.

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-019-12187-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761184PMC
September 2019

Genetic diversity in alveolar soft part sarcoma: A subset contain variant fusion genes, highlighting broader molecular kinship with other MiT family tumors.

Genes Chromosomes Cancer 2019 Aug 21. Epub 2019 Aug 21.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York.

Alveolar soft part sarcoma (ASPS) is a rare malignancy that, since its initial description, remains a neoplasm of uncertain histogenesis. The disease-defining molecular event characterizing the diagnosis of ASPS is the ASPSCR1-TFE3 fusion gene. Following identification of an index case of ASPS with a novel TFE3 fusion partner, we performed a retrospective review to determine whether this represents an isolated event. We identified two additional cases, for a total of three cases lacking ASPSCR1 partners. The average patient age was 46 years (range, 17-65); two patients were female. The sites of origin included the transverse colon, foot, and dura. Each case exhibited a histomorphology typical of ASPS, and immunohistochemistry was positive for TFE3 in all cases. Routine molecular testing of the index patient demonstrated a HNRNPH3-TFE3 gene fusion; the remaining cases were found to have DVL2-TFE3 or PRCC-TFE3 fusion products. The latter two fusions have previously been identified in renal cell carcinoma; to our knowledge, this is the first report of a HNRNPH3-TFE3 gene fusion. These findings highlight a heretofore underrecognized genetic diversity in ASPS, which appears to more broadly molecularly overlap with that of translocation-associated renal cell carcinoma and PEComa. These results have immediate implications in the diagnosis of ASPS since assays reliant upon ASPSCR1 may yield a false negative result. While these findings further understanding of the molecular pathogenesis of ASPS, issues related to the histogenesis of this unusual neoplasm remain unresolved.
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http://dx.doi.org/10.1002/gcc.22803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057290PMC
August 2019

Masked hypodiploidy: Hypodiploid acute lymphoblastic leukemia (ALL) mimicking hyperdiploid ALL in children: A report from the Children's Oncology Group.

Cancer Genet 2019 10 30;238:62-68. Epub 2019 Jul 30.

Department of Pathology, The Ohio State University, Columbus, OH, USA.

Hyperdiploidy with greater than 50 chromosomes is usually associated with favorable prognosis in pediatric acute lymphoblastic leukemia (ALL), whereas hypodiploidy with ≤43 chromosomes is associated with extremely poor prognosis. Sometimes, hypodiploidy is "masked" and patients do not have a karyotypically visible clone with ≤43 chromosomes. Instead, their abnormal karyotypes contain 50-78 or more chromosomes from doubling of previously hypodiploid cells. When the hypodiploid and doubled hyperdiploid clones are both present, patients can be identified by traditional test methods [karyotype, DNA Index (DI), fluorescence in situ hybridization (FISH)], but the incidence of masked hypodiploid cases in which only the doubled clone is visible is unknown. We analyzed 7013 patients with B-ALL enrolled in COG AALL03B1 (2003-2011) for whom chromosome studies were available. Of 115 patients with hypodiploidy (25-39 chromosomes), karyotypes of 40 showed only the hypodiploid clone, 47 showed mosaicism with both hypodiploid and hyperdiploid (doubled) karyotypes, and 28 with masked hypodiploidy showed only a hyperdiploid (doubled) clone. Unique karyotypic signatures were identified, and widespread loss of heterozygosity (LOH) was seen in the microsatellite panel for all patients with masked hypodiploidy. An increased awareness of the unusual karyotypic profile associated with a doubled hypodiploid clone and coordinated use of DI, FISH, and LOH studies when indicated can identify patients with masked hypodiploidy and allow appropriate treatment selection.
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http://dx.doi.org/10.1016/j.cancergen.2019.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768693PMC
October 2019

An aggressive central giant cell granuloma in a pediatric patient: case report and review of literature.

J Otolaryngol Head Neck Surg 2019 Jul 18;48(1):32. Epub 2019 Jul 18.

Department of Otolaryngology - Head and Neck Surgery, Faculty of Medicine, University of Ottawa, Ottawa, Canada.

Background: Central giant cell granulomas are benign tumours of the mandible, presenting in children and young adults. Divided into non- and aggressive subtypes, the aggressive subtype is relatively rare and can occasionally progress rapidly, resulting in significant morbidity.

Case Presentation: We present a case of an aggressive central giant cell granuloma (CGCG) in a six year-old female. The lesion originated in the right mandibular ramus and progressed rapidly to involve the condyle. Diagnosis was made using a combination of imaging and pathology. A timely en bloc resection of the hemi-mandible was performed with placement of a reconstructive titanium plate and condylar prosthesis.

Conclusion: Our case demonstrates the importance of considering CGCG in the differential diagnosis of rapidly progressive mandibular lesions in the pediatric population. Prompt diagnosis and management can greatly improve long-term outcomes.
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http://dx.doi.org/10.1186/s40463-019-0356-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637537PMC
July 2019

Immunohistochemistry for ATRX Can Miss ATRX Mutations: Lessons From Neuroblastoma.

Am J Surg Pathol 2019 09;43(9):1203-1211

Division of Pathology.

Neuroblastoma is the most common extracranial solid tumor of childhood with a median age of presentation of 17 months. A common theme in high-risk neuroblastoma is maintenance of telomeres, one mechanism for which involves alternate lengthening of telomeres (ALT) associated with ATRX gene mutations. Mutations are believed to result in loss of ATRX protein, and therefore immunohistochemistry is used to detect mutations. We screened 133 cases of neuroblastoma by ATRX immunohistochemistry, and found 9 cases with partial to total absence of ATRX. Sequencing for ATRX mutations detected a mutation in 1 of 9 cases, suggesting immunostaining was not reliable for detecting mutations. To correlate immunostaining with ALT, fluorescence in situ hybridization (FISH) for ALT was performed in 6 of these cases and 5 (from 4 patients) showed ALT, implying impaired ATRX protein function, despite the failure to identify a mutation. Two other cases with large deletions in the ATRX gene showed diffusely positive staining for ATRX protein but showed ALT by FISH. Four of the 6 patients with ALT-positive tumors were over 5 years old. Therefore, 29 additional patients 5 years old and above with ATRX-positive tumors were screened for ALT by FISH and 6 additional cases with ALT were detected, bringing the total to 29% (10/34) of children 5 years old and above, 70% of which showed positive ATRX immunohistochemistry. Patients with ATRX mutations in neuroblastoma tend to have a more chronic and progressive course of disease. Screening neuroblastoma tumors at diagnosis for ATRX mutations may help identify patients who might benefit from personalized therapy directed against ALT. However, relaying on negative immunohistochemistry for ATRX protein to identify ALT in neuroblastoma may miss a significant proportion of patients. The addition of FISH for ALT as part of the diagnostic workup, especially for older children (5 y old and above), would help ensure that patients are correctly identified for anti-ALT therapy.
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http://dx.doi.org/10.1097/PAS.0000000000001322DOI Listing
September 2019

MYCN Amplified Relapse Following Resolution of MYCN Nonamplified 4S Neuroblastoma With Placental Involvement: A Case Report and Review of the Literature.

J Pediatr Hematol Oncol 2019 Jul;41(5):388-391

Departments of Haematology/Oncology, Division of Paediatrics.

Congenital neuroblastoma with placental involvement is exceptionally rare, but mortality is high. Detailed examination of placenta including MYCN amplification and segmental chromosomal aberrations should be performed in all suspected cases, as it is noninvasive and readily available. Maternal dissemination has not been reported. In this manuscript, we describe an infant with placental diagnosis of MYCN nonamplified congenital neuroblastoma. This is the first report of a recurrence of congenital 4S neuroblastoma following resolution in which MYCN amplification is only detected in the recurrence. Germline sequencing using a large comprehensive cancer panel did not reveal variants in candidate cancer predisposition genes.
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http://dx.doi.org/10.1097/MPH.0000000000001515DOI Listing
July 2019

Aggressive embryonal rhabdomyosarcoma in a 3-month-old boy: A clinical and molecular analysis.

Pediatr Hematol Oncol 2018 Oct - Nov;35(7-8):407-414. Epub 2019 Feb 26.

a Division of Haematology/Oncology, The Hospital for Sick Children, Department of Pediatrics , University of Toronto , Toronto , ON , Canada.

Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in the pediatric age group. While RMS has been traditionally classified on the basis of its histological appearance (with embryonal and alveolar being most common), it is now clear that the PAX-FOXO1 fusion product drives prognosis. We report here a case of pelvic embryonal RMS in a 3-month-old male who was subsequently found to have developed brain metastases during the course of chemotherapy. Cytogenetic analysis of the brain metastases at the time of autopsy as well as next-generation sequencing analysis revealed a reciprocal translocation involving the SH3 domain containing ring finger 3 gene (SH3RF3, on chromosome 2q13) and the Lipase C gene (LIPC, on chromosome 15q21.3). Due to the poor quality of the pretreatment and postresection samples, cytogenetics and NGS analysis looking for the presence of this balanced translocation in these specimens could not be performed. To the authors' knowledge, this translocation has never been described in RMS. Further studies are needed to determine the biological and clinical implications of this novel translocation.
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http://dx.doi.org/10.1080/08880018.2019.1569185DOI Listing
April 2019

Rearrangement bursts generate canonical gene fusions in bone and soft tissue tumors.

Science 2018 08;361(6405)

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, UK.

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between , a gene encoding an RNA binding protein, and E26 transformation-specific (ETS) transcription factors. We explored how and when fusions arise by studying the whole genomes of Ewing sarcomas. In 52 of 124 (42%) of tumors, the fusion gene arises by a sudden burst of complex, loop-like rearrangements, a process called chromoplexy, rather than by simple reciprocal translocations. These loops always contained the disease-defining fusion at the center, but they disrupted multiple additional genes. The loops occurred preferentially in early replicating and transcriptionally active genomic regions. Similar loops forming canonical fusions were found in three other sarcoma types. Chromoplexy-generated fusions appear to be associated with an aggressive form of Ewing sarcoma. These loops arise early, giving rise to both primary and relapse Ewing sarcoma tumors, which can continue to evolve in parallel.
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http://dx.doi.org/10.1126/science.aam8419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176908PMC
August 2018

Clinicopathologic Features of a Series of Primary Renal CIC-rearranged Sarcomas With Comprehensive Molecular Analysis.

Am J Surg Pathol 2018 10;42(10):1360-1369

Alpert Medical School of Brown University, Rhode Island Hospital, Providence, RI.

CIC-rearranged sarcomas rarely occur in visceral organs including the kidney. The most common fusion partner with CIC is the DUX4 gene, but variant fusion partners have also been reported. Herein, we describe the clinicopathologic features and comprehensive molecular profiling of 4 cases of primary renal CIC-rearranged sarcomas. All cases occurred in females, age range 13 to 82 years and included 3 resections and 1 needle biopsy specimen. There was a tendency for development of metastatic disease predominantly to the lungs and poor disease outcome despite different treatment strategies. Histologically, variable round cell (20% to 100%), spindle cell (0% to 80%), and rhabdoid morphologies (0% to 20%) were seen. By immunohistochemistry diffuse WT1 nuclear (2 to 3+, ∼90%) labeling was present in 1 case, with cytoplasmic staining in the others (3+, 40% to 75%). CD99 was focally positive in all 4 cases (≤10%); 1 case each was diffusely positive for c-myc (2 to 3+, ∼90%) and ETV4 (3+, ∼90%); 1 case was focally positive for c-myc (2+, ∼5%) and calretinin (2+, ∼5%); and all cases were negative for cytokeratin and NKX2.2. CIC rearrangement by fluorescence in situ hybridization was present in the 3 cases tested. Comprehensive genomic profiling (CGP) of 3 cases revealed a CIC-DUX4 fusion in 2 cases, and 1 CIC-NUTM1 fusion. All 4 CIC-rearranged renal sarcomas had low mutation burden, and except HLA-A and MLL mutations lacked genomic alterations in other oncogenic drivers. Material from the needle biopsy was insufficient for CGP but that case was positive with the DUX4 immunohistochemical stain as were the 2 CIC-DUX4 tumors. In conclusion, CIC-rearranged sarcomas rarely occur in the kidney with a tendency for poor outcome and in this series we illustrate an example with CIC-NUTM1 fusion, an emerging variant, at a visceral site. Testing by fluorescence in situ hybridization or CGP is optimal to avoid missing cases that harbor variant fusion partners.
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http://dx.doi.org/10.1097/PAS.0000000000001098DOI Listing
October 2018

Fluorescent In Situ Hybridization for TP53 in the Diagnosis of Pediatric Osteogenic Sarcoma.

Am J Surg Pathol 2018 06;42(6):744-749

Division of Pathology.

Osteogenic sarcoma (OS) is the most common malignant bone tumor in children and adolescents. Despite advances in molecular genetic characterization of pediatric and adult tumors, the diagnosis of OS still depends almost entirely on light microscopy. The lack of consistent genetic changes in OS has greatly hindered the development of any diagnostic molecular test. Recently, whole-genome sequencing has shown that ~50% of cases of OS have a translocation involving the TP53 gene with breakpoints confined to the first intron. We developed a 2 color break-apart fluorescent in situ hybridization (FISH) probe for intron 1 of TP53 and applied it to an archived series to assess its diagnostic utility. The study group included 37 cases of OS (including osteoblastic, chondroblastic, and fibroblastic), as well as 53 cases of non-OS pediatric sarcomas (including Ewing sarcoma, rhabdomyosarcoma, undifferentiated small cell sarcoma, CCNB3-BCOR sarcoma, CIC-DUX sarcoma, synovial sarcoma, and malignant peripheral nerve sheath tumor) and 27 cases of benign bone lesions (including osteoblastoma, chondromyxoid fibroma, fibrous dysplasia, and fibro-osseous dysplasia). A rearranged signal was found in 20/37 cases (54%) of OS and in none of the other sarcomas or benign bone lesions, giving the FISH test 100% specificity for a diagnosis of OS. p53 immunostaining was generally not predictive of the results obtained by FISH and could not substitute for this test. This FISH probe offers a simple and specific genetic test to aid in the diagnosis of OS, despite the genetic complexity of this tumor.
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http://dx.doi.org/10.1097/PAS.0000000000001054DOI Listing
June 2018

Multiplex Detection of Pediatric Low-Grade Glioma Signature Fusion Transcripts and Duplications Using the NanoString nCounter System.

J Neuropathol Exp Neurol 2017 Jul;76(7):562-570

Department of Cancer and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Divisions of Pathology and Genome Diagnostics, Department of Paediatric Laboratory Medicine; and Division of Hematology and Oncology, The Division of Hematology and Oncology should be followed by Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada. Hospital for Sick Children, Toronto, Ontario, Canada; Department of Radiation Oncology, Tata Memorial Hospital, Parel, Mumbai, India; and Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

Previous studies identified recurrent fusion and duplication events in pediatric low-grade glioma (pLGG). In addition to their role in diagnosis, the presence of these events aid in dictating therapy and predicting patient survival. Clinically, BRAF alterations are most commonly identified using fluorescent in situ hybridization (FISH). However, this method is costly, labor-intensive and does not identify nonBRAF events. Here, we evaluated the NanoString nCounter gene expression system for detecting 32 of the most commonly reported fusion/duplication events in pLGG. The assay was validated on 90 pLGG samples using FISH as the gold standard and showed sensitivity and specificity of 97% and 98%, respectively. We next profiled formalin-fixed paraffin-embedded preserved biopsy specimens from 429 pLGG cases. 171 (40%) of the cases within our cohort tested positive for a fusion or duplication event contained within our panel. These events, in order of prevalence, were KIAA1549-BRAF 16;9 (89/171, 52.0%), KIAA1549-BRAF 15;9 (42/171, 24.6%), KIAA1549-BRAF 16;11 (14/171, 8.2%), FGFR1-TACC1 17;7 (13/171, 7.6%), MYBL1 duplication (5/171, 2.9%), KIAA1549-BRAF 18;10 (4/171, 2.3%), KIAA1549-BRAF 15;11 (2/171, 1.2%), FAM131B-BRAF 2;9 (1/171, 0.6%), and RNF130-BRAF 3;9 (1/171, 0.6%). This work introduces NanoString as a viable clinical replacement for the detection of fusion and duplication events in pLGG.
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http://dx.doi.org/10.1093/jnen/nlx042DOI Listing
July 2017

The clinical impact of copy number variants in inherited bone marrow failure syndromes.

NPJ Genom Med 2017 May;2

Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, ON, Canada.

Inherited bone marrow failure syndromes (IBMFSs) comprise a genetically heterogeneous group of diseases with hematopoietic failure and a wide array of physical malformations. Copy number variants (CNVs) were reported in some IBMFSs. It is unclear what impact CNVs play in patients evaluated for a suspected diagnosis of IBMFS. Clinical and genetic data of 323 patients from the Canadian Inherited Marrow Failure Registry from 2001 to 2014, who had a documented genetic work-up, were analyzed. Cases with pathogenic CNVs (at least 1 kilobasepairs) were compared to cases with other mutations. Genotype-phenotype correlations were performed to assess the impact of CNVs. Pathogenic nucleotide-level mutations were found in 157 of 303 tested patients (51.8%). Genome-wide CNV analysis by single nucleotide polymorphism arrays or comparative genomic hybridization arrays revealed pathogenic CNVs in 11 of 67 patients tested (16.4%). In four of these patients, identification of CNV was crucial for establishing the correct diagnosis as their clinical presentation was ambiguous. Eight additional patients were identified to harbor pathogenic CNVs by other methods. Of the 19 patients with pathogenic CNVs, four had compound-heterozygosity of a CNV with a nucleotide-level mutation. Pathogenic CNVs were associated with more extensive non-hematological organ system involvement (=0.0006), developmental delay (=0.006) and short stature (=0.04) compared to nucleotide-level mutations. In conclusion, a significant proportion of patients with IBMFSs harbor pathogenic CNVs which were associated with a more extensive non-hematological phenotype in this cohort. Patients with a phenotype suggestive of IBMFSs but without identification of pathogenic nucleotide-level mutations should undergo specific testing for CNVs.
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http://dx.doi.org/10.1038/s41525-017-0019-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498150PMC
May 2017

Recurrent Cytogenetic Abnormalities in Acute Lymphoblastic Leukemia.

Authors:
Mary Shago

Methods Mol Biol 2017 ;1541:257-278

Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada, M5G 1X8.

Both B-cell and T-cell acute lymphoblastic leukemia (ALL) exhibit recurrent cytogenetic alterations, many with prognostic implications. This chapter overviews the major recurrent categories of cytogenetic abnormalities associated with ALL, with an emphasis on the detection and characterization of these cases by G-band and FISH analyses.
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http://dx.doi.org/10.1007/978-1-4939-6703-2_21DOI Listing
January 2018

Chromosome Preparation for Acute Lymphoblastic Leukemia.

Authors:
Mary Shago

Methods Mol Biol 2017 ;1541:19-31

Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada, M5G 1X8.

The chromosome abnormalities observed in acute lymphoblastic leukemia have been demonstrated to contribute to patient management and treatment stratification. This chapter provides a basic protocol for the procurement, culture, harvest, and slide preparation of bone marrow aspirate and unstimulated peripheral blood specimens.
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http://dx.doi.org/10.1007/978-1-4939-6703-2_3DOI Listing
January 2018

TFE3-positive renal cell carcinomas are not always Xp11 translocation carcinomas: Report of a case with a TPM3-ALK translocation.

Pathol Res Pract 2016 Oct 12;212(10):937-942. Epub 2016 Jul 12.

Division of Pathology, Hospital for Sick Children, Toronto, Canada; Department of Pathobiology and Laboratory Medicine, University of Toronto, Toronto, Canada.

Translocation-associated renal cell carcinoma (RCC) is a distinct subtype of RCC with gene rearrangements of the TFE3 or TFEB loci. The TFE3 gene is located at Xp11 and can fuse to a number of translocation partners, resulting in high nuclear expression of TFE3 protein. TFE3 immunostaining is often used as a surrogate marker for a TFE3 translocation. We report a case of an RCC that expressed TFE3 but showed only gain of TFE3 rather than a translocation. Moreover, this case had a t(1;2) translocation fusing ALK and TMP3, identical to that seen in inflammatory myofibroblastic tumour. There was resulting overexpression of ALK protein in a cytoplasmic and membranous pattern. The patient was not treated with chemotherapy but following regional nodal recurrence, an ALK inhibitor was added and the patient remains alive one year later. There are only rare reports of RCC with an ALK-TMP3 fusion, and these tumours can express TFE3 on some unknown basis not related to a TFE3 translocation. Any RCC positive for TFE3 and lacking a translocation should be tested for ALK expression and translocation. Recognition of this subtype of RCC will allow ALK inhibitor therapy to be added, in the hope of improving patient outcome.
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http://dx.doi.org/10.1016/j.prp.2016.07.004DOI Listing
October 2016

Frequency and outcome of pediatric acute lymphoblastic leukemia with ZNF384 gene rearrangements including a novel translocation resulting in an ARID1B/ZNF384 gene fusion.

Pediatr Blood Cancer 2016 11 8;63(11):1915-21. Epub 2016 Jul 8.

Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada.

Background: ZNF384 gene rearrangements with multiple partner genes are recurrent in acute leukemia and are most often associated with a precursor B cell immunophenotype. The overall incidence of this genetic category of leukemia is uncertain.

Procedure: Patients with ZNF384 gene rearrangements from a cohort of 240 precursor B cell acute lymphoblastic leukemia (ALL) pediatric patients over a 3.5-year time period were characterized with detailed cytogenetic, FISH, genomic, and clinical analyses.

Results: Seven of the 240 patients were identified to have ZNF384 gene rearrangements including partner genes TCF3 (four patients), EWSR1 (one patient), EP300 (one patient), and the novel gene partner ARID1B (one patient). The translocations were confirmed by FISH analysis and with RNA sequencing for the EP300 and ARID1B partner genes. Genomic microarray analysis showed an average of 2.7 copy number alterations in each case with no evidence of imbalance at the translocation breakpoints. Six of the patients with ZNF384 gene rearrangements had precursor B cell ALL with a CD10- immunophenotype and myeloid-associated antigens. One of the patients also had myeloperoxidase expression and was diagnosed as mixed phenotype B/myeloid acute leukemia. None of the patients have relapsed with event-free survival ranging from 6 years 2 months to 9 years 2 months.

Conclusions: This study suggests that the frequency of ZNF384 gene rearrangement in pediatric precursor B cell ALL is approximately 3%. The ARID1B gene, commonly mutated in multiple types of cancer, was identified as an additional ZNF384 gene fusion partner.
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http://dx.doi.org/10.1002/pbc.26116DOI Listing
November 2016

Indexing Effects of Copy Number Variation on Genes Involved in Developmental Delay.

Sci Rep 2016 07 1;6:28663. Epub 2016 Jul 1.

The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada.

A challenge in clinical genomics is to predict whether copy number variation (CNV) affecting a gene or multiple genes will manifest as disease. Increasing recognition of gene dosage effects in neurodevelopmental disorders prompted us to develop a computational approach based on critical-exon (highly expressed in brain, highly conserved) examination for potential etiologic effects. Using a large CNV dataset, our updated analyses revealed significant (P < 1.64 × 10(-15)) enrichment of critical-exons within rare CNVs in cases compared to controls. Separately, we used a weighted gene co-expression network analysis (WGCNA) to construct an unbiased protein module from prenatal and adult tissues and found it significantly enriched for critical exons in prenatal (P < 1.15 × 10(-50), OR = 2.11) and adult (P < 6.03 × 10(-18), OR = 1.55) tissues. WGCNA yielded 1,206 proteins for which we prioritized the corresponding genes as likely to have a role in neurodevelopmental disorders. We compared the gene lists obtained from critical-exon and WGCNA analysis and found 438 candidate genes associated with CNVs annotated as pathogenic, or as variants of uncertain significance (VOUS), from among 10,619 developmental delay cases. We identified genes containing CNVs previously considered to be VOUS to be new candidate genes for neurodevelopmental disorders (GIT1, MVB12B and PPP1R9A) demonstrating the utility of this strategy to index the clinical effects of CNVs.
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http://dx.doi.org/10.1038/srep28663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929460PMC
July 2016

Primary Undifferentiated Sarcoma of the Kidney Harboring a Novel Variant of CIC-DUX4 Gene Fusion.

Am J Surg Pathol 2016 09;40(9):1298-301

*Department of Pathology, Rhode Island Hospital & Alpert Medical School of Brown University, Providence, RI †Department of Pathology, The Hospital for Sick Children & University of Toronto, Toronto, ON, Canada ‡Foundation Medicine Inc. Cambridge, MA.

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http://dx.doi.org/10.1097/PAS.0000000000000688DOI Listing
September 2016

TFE3-Expressing Perivascular Epithelioid Cell Neoplasm (PEComa) of the Sella Turcica.

Endocr Pathol 2017 Mar;28(1):22-26

Department of Pathology, University Health Network, Toronto, ON, Canada.

We report a primary central nervous system (CNS) perivascular epithelioid cell tumor (PEComa) in a middle-aged female patient. The tumor occurred in suprasellar location with secondary extension into the sella turcica. The patient presented with intracranial hemorrhage and an altered level of consciousness. The tumor had morphologic features matching those of other previously described TFE3-translocated PEComas, including epithelioid morphology, diffuse and strong nuclear immunoreactivity for TFE3, and minimal staining with myoid markers. The TFE3 break-apart FISH testing showed a slight splitting of one of the TFE3 signals in 49.5 % of nuclei. This case illustrates that PEComas should be added to the growing list of mesenchymal tumors that can be encountered in the CNS and specifically in the vicinity of the pituitary gland. The recognition of this entity is of significance given their underlying pathogenesis and possible management implications.
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http://dx.doi.org/10.1007/s12022-016-9434-7DOI Listing
March 2017

Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN).

J Med Genet 2015 Nov 4;52(11):738-48. Epub 2015 Sep 4.

Genetics and Genome Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada.

Background: Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN.

Methods: We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease.

Results: We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease.

Conclusions: Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.
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http://dx.doi.org/10.1136/jmedgenet-2015-103292DOI Listing
November 2015

The impact of category, cytopathology and cytogenetics on development and progression of clonal and malignant myeloid transformation in inherited bone marrow failure syndromes.

Haematologica 2015 May 14;100(5):633-42. Epub 2015 Feb 14.

Marrow Failure and Myelodysplasia Program, Division of Haematology/Oncology, Department of Paediatrics and the Genetics and Genome Biology Program, Research Institute, The Hospital for Sick Children and the University of Toronto, Ontario, Canada

Inherited bone marrow failure syndromes are a group of rare, heterogeneous genetic disorders with a risk of clonal and malignant myeloid transformation including clonal marrow cytogenetic abnormalities, myelodysplastic syndrome and acute myeloid leukemia. The clinical characteristics, risk classification, prognostic factors and outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes are largely unknown. The aims of this study were to determine the impact of category, cytopathology and cytogenetics, the three components of the "Category Cytology Cytogenetics" classification of pediatric myelodysplastic syndrome, on the outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure. We used data from the Canadian Inherited Marrow Failure Registry. Among 327 patients with inherited bone marrow failure syndrome enrolled in the registry, the estimated risk of clonal and malignant myeloid transformation by the age of 18 years was 37%. The risk of clonal and malignant myeloid transformation varied according to the type of inherited bone marrow failure syndrome but was highest in Fanconi anemia. The development of clonal and malignant myeloid transformation significantly affected overall survival. Mortality varied based on cytopathological group. The largest group of patients had refractory cytopenia. Clonal marrow cytogenetic abnormalities were identified in 87% of patients with clonal and malignant myeloid transformation, and different cytogenetic groups had different impacts on disease progression. We conclude that category, cytopathology and cytogenetics in cases of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes have an important impact on outcome and that the classification of such cases should incorporate these factors.
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http://dx.doi.org/10.3324/haematol.2014.117457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420212PMC
May 2015

BRAF mutation and CDKN2A deletion define a clinically distinct subgroup of childhood secondary high-grade glioma.

J Clin Oncol 2015 Mar 9;33(9):1015-22. Epub 2015 Feb 9.

Matthew Mistry, Nataliya Zhukova, Daniele Merico, Rahul Krishnatry, Mary Shago, James Stavropoulos, Noa Alon, Peter N. Ray, Vilma Navickiene, Joshua Mangerel, Marc Remke, Vijay Ramaswamy, Ana Guerreiro Stucklin, Martin Li, Edwin J. Young, Cindy Zhang, Pedro Castelo-Branco, Doua Bakry, Suzanne Laughlin, James T. Rutka, Peter B. Dirks, Michael D. Taylor, Mark Greenberg, David Malkin, Annie Huang, Eric Bouffet, Cynthia E. Hawkins, and Uri Tabori; The Hospital for Sick Children; Matthew Mistry, Patricia Rakopoulos, Rahul Krishnatry, Joshua Mangerel, Pawel Buczkowicz, Ana Guerreiro Stucklin, Doua Bakry, Adam Shlien, Mark Greenberg, David Malkin, Annie Huang, Eric Bouffet, Cynthia E. Hawkins, and Uri Tabori, University of Toronto; Jason D. Pole, Pediatric Oncology Group of Ontario, Toronto, Ontario; Jennifer Chan, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada; Pedro Castelo-Branco, Universidade do Algarve, Faro, Portugal; Keith L. Ligon, Dana-Farber/Boston Children's Cancer Center, Boston, MA.

Purpose: To uncover the genetic events leading to transformation of pediatric low-grade glioma (PLGG) to secondary high-grade glioma (sHGG).

Patients And Methods: We retrospectively identified patients with sHGG from a population-based cohort of 886 patients with PLGG with long clinical follow-up. Exome sequencing and array CGH were performed on available samples followed by detailed genetic analysis of the entire sHGG cohort. Clinical and outcome data of genetically distinct subgroups were obtained.

Results: sHGG was observed in 2.9% of PLGGs (26 of 886 patients). Patients with sHGG had a high frequency of nonsilent somatic mutations compared with patients with primary pediatric high-grade glioma (HGG; median, 25 mutations per exome; P = .0042). Alterations in chromatin-modifying genes and telomere-maintenance pathways were commonly observed, whereas no sHGG harbored the BRAF-KIAA1549 fusion. The most recurrent alterations were BRAF V600E and CDKN2A deletion in 39% and 57% of sHGGs, respectively. Importantly, all BRAF V600E and 80% of CDKN2A alterations could be traced back to their PLGG counterparts. BRAF V600E distinguished sHGG from primary HGG (P = .0023), whereas BRAF and CDKN2A alterations were less commonly observed in PLGG that did not transform (P < .001 and P < .001 respectively). PLGGs with BRAF mutations had longer latency to transformation than wild-type PLGG (median, 6.65 years [range, 3.5 to 20.3 years] v 1.59 years [range, 0.32 to 15.9 years], respectively; P = .0389). Furthermore, 5-year overall survival was 75% ± 15% and 29% ± 12% for children with BRAF mutant and wild-type tumors, respectively (P = .024).

Conclusion: BRAF V600E mutations and CDKN2A deletions constitute a clinically distinct subtype of sHGG. The prolonged course to transformation for BRAF V600E PLGGs provides an opportunity for surgical interventions, surveillance, and targeted therapies to mitigate the outcome of sHGG.
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http://dx.doi.org/10.1200/JCO.2014.58.3922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356711PMC
March 2015

CNS-PNETs with C19MC amplification and/or LIN28 expression comprise a distinct histogenetic diagnostic and therapeutic entity.

Acta Neuropathol 2014 Aug 20;128(2):291-303. Epub 2014 May 20.

Division of Hematology-Oncology, Department of Pediatrics, The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumor Research Centre, Peter Gilgan CRL,686 Bay Street, 17th Floor, 179712, Toronto, ON, M5G0A4, Canada.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.
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http://dx.doi.org/10.1007/s00401-014-1291-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159569PMC
August 2014

Mosaic microdeletion of 17p11.2-p12 and duplication of 17q22-q24 in a girl with Smith-Magenis phenotype and peripheral neuropathy.

Am J Med Genet A 2014 Mar 19;164A(3):748-52. Epub 2013 Dec 19.

Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, Canada.

We report on a girl with a de novo mosaic derivative chromosome 17 involving a 7.4 Mb deletion of chromosome region 17p11.2 to 17p12 and a duplication of a 12.35 Mb region at 17q22 to 17q24. She was ascertained because of developmental delay, peripheral neuropathy, brachydactyly and minor anomalies. The derivative chromosome was present in approximately 12% of lymphocytes based on FISH studies, and was detected by array comparative genomic hybridization. To our knowledge, this is the third case of mosaicism involving deletion of the 17p11.2 region and the lowest level of mosaicism reported in a patient with Smith-Magenis syndrome (SMS).
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http://dx.doi.org/10.1002/ajmg.a.36322DOI Listing
March 2014