ROS1 Alterations as a Potential Driver of Gliomas in Infant, Pediatric, and Adult Patients.

Gliomas harboring oncogenic ROS1 alterations are uncommon and primarily described in infants. Our goal was to characterize the clinicopathological features and molecular signatures of the full spectrum of ROS1 fusion-positive gliomas across all age groups. Through a retrospective multi-institutional collaboration, we report a collection of unpublished ROS1 fusion gliomas along with the characterization and meta-analysis of new and published cases. A cohort of 32 new and 58 published cases was divided into the following 3 age groups: 19 infants, 40 pediatric patients, and 31 adults with gliomas. Tumors in infants and adults showed uniformly high-grade morphology; however, tumors in pediatric patients exhibited diverse histologic features. The GOPC::ROS1 fusion was prevalent (61/79, 77%) across all age groups, and 10 other partner genes were identified. Adult tumors showed recurrent genomic alterations characteristic of IDH wild-type glioblastoma, including the +7/-10/CDKN2A deletion; amplification of CDK4, MDM2, and PDGFRA genes; and mutations involving TERTp, TP53, PIK3R1, PIK3CA, PTEN, and NF1 genes. Infant tumors showed few genomic alterations, whereas pediatric tumors showed moderate genomic complexity. The outcomes were significantly poorer in adult patients. Although not statistically significant, tumors in infant and pediatric patients with high-grade histology and in hemispheric locations appeared more aggressive than tumors with lower grade histology or those in nonhemispheric locations. In conclusion, this study is the largest to date to characterize the clinicopathological and molecular signatures of ROS1 fusion-positive gliomas from infant, pediatric, and adult patients. We conclude that ROS1 likely acts as a driver in infant and pediatric gliomas and as a driver or codriver in adult gliomas. Integrated comprehensive clinical testing might be helpful in identifying such patients for possible targeted therapy.

Integrated genetic profiling of archival pediatric high-grade glial tumors and reassessment with 2021 WHO classification of paediatric CNS tumours.

Though rare, pediatric high-grade gliomas (pHGG) are a leading cause of cancer-related mortality in children. We wanted to determine whether our currently available clinical laboratory methods could better define diagnosis for pHGG that had been archived at our institution for the past 20 years (1998 to 2017). We investigated 33 formalin-fixed paraffin-embedded pHGG using ThermoFisher Oncoscan SNP microarray with somatic mutation analysis, Sanger sequencing, and whole genome sequencing. These data were correlated with historical histopathological, chromosomal, clinical, and radiological data. Tumors were subsequently classified according to the 2021 WHO Classification of Paediatric CNS Tumours. All 33 tumors were found to have genetic aberrations that placed them within a 2021 WHO subtype and/or provided prognostic information; 6 tumors were upgraded from WHO CNS grade 3 to grade 4. New pHGG genetic features were found including two small cell glioblastomas with H3 G34 mutations not previously described; one tumor with STRN-NTRK2 fusion; and a congenital diffuse leptomeningeal glioneuronal tumor without a chromosomal 1p deletion but with KIAA1549-BRAF fusion. Overall, the combination of laboratory methods yielded key information for tumor classification. Thus, even small studies of these uncommon tumor types may yield new genetic features and possible new subtypes that warrant future investigations.

Chromosomal microarray analysis, including constitutional and neoplastic disease applications, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Chromosomal microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism array, are widely applied in the diagnostic evaluation for both constitutional and neoplastic disorders. In a constitutional setting, this technology is accepted as the first-tier test for the evaluation of chromosomal imbalances associated with intellectual disability, autism, and/or multiple congenital anomalies. Furthermore, chromosomal microarray analysis is recommended for patients undergoing invasive prenatal diagnosis with one or more major fetal structural abnormalities identified by ultrasonographic examination, and in the evaluation of intrauterine fetal demise or stillbirth when further cytogenetic analysis is desired. This technology also provides important genomic data in the diagnosis, prognosis, and therapy of neoplastic disorders, including both hematologic malignancies and solid tumors. To assist clinical laboratories in the validation of chromosomal microarray methodologies for constitutional and neoplastic applications, the American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has developed these updated technical laboratory standards, which replace the ACMG technical standards and guidelines for microarray analysis in constitutional and neoplastic disorders previously published in 2013.

Factors Affecting Migration to GRCh38 in Laboratories Performing Clinical Next-Generation Sequencing.

The most recent build of the human reference genome, GRCh38, was released in 2013. However, many laboratories performing next-generation sequencing (NGS) continue to align to GRCh37. Our aim was to assess the number of clinical diagnostic laboratories that have migrated to GRCh38 and discern factors impeding migration for those still using GRCh37. A brief, five-question survey was electronically administered to 71 clinical laboratories offering constitutional NGS-based testing and analyzed categorically. Twenty-eight responses meeting inclusion criteria were collected from 24 academic and four commercial diagnostic laboratories. Most of these (14; 50%) reported volumes of <500 NGS-based tests in 2019. Only two respondents (7%) had already migrated entirely to GRCh38; most laboratories (15; 54%) had no plans to migrate. The two prevailing reasons for not yet migrating were as follows: laboratories did not feel the benefits outweighed the time and monetary costs (14; 50%); and laboratories had insufficient staff to facilitate the migration (12; 43%). These data, although limited, suggest most clinical molecular laboratories are reluctant to migrate to GRCh38, and there appear to be multiple obstacles to overcome before GRCh38 is widely adopted.

Clinical Validation of Somatic Mutation Detection by the OncoScan CNV Plus Assay.

The OncoScan CNV Plus Assay (OS+) is a single-nucleotide polymorphism microarray platform that can detect 74 hotspot somatic mutations (SMs) in nine genes via molecular inversion probes. We report validation of the SM component of OS+ using a cohort of pediatric high-grade brain tumor specimens. SM calls were generated from 46 brain tumor cases, most tested orthogonally via bidirectional Sanger sequencing. The initial calling algorithm result showed that 31 tumors were positive and 15 were negative for SM, with a total of 71 OS+ SM calls [28 high-confidence (HC) and 43 low-confidence (LC)]. Sanger sequencing was performed for 54 of the 71 calls (27 HC and 27 LC), as well as for 21 randomly selected hotspots across the 15 OS+ negative cases. HC calls (except EGFR) Sanger sequencing confirmed positive, negative calls confirmed negative, but none of the LC calls were Sanger-confirmed positive. An update of the OS+ algorithm resolved the LC calls, but of the 11 HC SM EGFR calls, Sanger sequencing confirmed only one. Two PTEN SM calls by OS+ in two separate cases were also negative per Sanger sequencing. We conclude that a majority of HC OS+ SM calls were accurate, except calls identified in EGFR and PTEN. Clinically, we report SMs identified by OS+ only after Sanger sequencing verification.

Application of 2016 WHO classification in the diagnosis of paediatric high-grade MYC-negative mature B-cell lymphoma with Burkitt-like morphological features.

AIMS: Historically, there has been no consensus on the diagnostic classification of high-grade B-cell lymphoma (HGBCL) with morphological features of Burkitt lymphoma (BL) but no MYC gene rearrangement (MYC-negative). The 2016 WHO classification of tumours of haematopoietic and lymphoid tissues has shed some light on this field with the modification of the grey-zone lymphoma with features intermediate between BL and diffuse large B-cell lymphoma, and the creation of several new entities. The aim of this study was to investigate how the revised WHO classification affects our practice in diagnosing these lymphomas in children. METHODS: We retrospectively reviewed cases of mature HGBCL diagnosed at our hospital between 2015 and 2018. RESULTS: Among 14 mature HGBCL cases with BL morphological features, 11 showed MYC rearrangement consistent with BL and 3 were MYC-negative. Two MYC-negative cases showed regions of 11q gain and loss by microarray consistent with Burkitt-like lymphoma with 11q aberration (BLL-11q). The third MYC-negative case showed diffuse and strong MUM1 expression, translocation involving 6p25 by chromosome analysis and IRF4 rearrangement by fluorescence in situ hybridisation analysis consistent with large B-cell lymphoma with IRF4 rearrangement (LBL-IRF4). All patients were treated according to applicable chemotherapeutic protocols and achieved remission. CONCLUSIONS: BLL-11q and LBL-IRF4, two newly defined entities, should be considered in paediatric MYC-negative mature HGBCL cases. Accurate diagnosis needs careful histopathological examination and proper cytogenetic testing. Since they have unique cytogenetic features, specific treatments for them may emerge in the future. Therefore, accurate diagnosis based on the 2016 WHO classification is clinically significant.

Near haploidization is a genomic hallmark which defines a molecular subgroup of giant cell glioblastoma.

BACKGROUND: Giant cell glioblastoma (gcGBM) is a rare histologic subtype of glioblastoma characterized by numerous bizarre multinucleate giant cells and increased reticulin deposition. Compared with conventional isocitrate dehydrogenase (IDH)-wildtype glioblastomas, gcGBMs typically occur in younger patients and are generally associated with an improved prognosis. Although prior studies of gcGBMs have shown enrichment of genetic events, such as TP53 alterations, no defining aberrations have been identified. The aim of this study was to evaluate the genomic profile of gcGBMs to facilitate more accurate diagnosis and prognostication for this entity. METHODS: Through a multi-institutional collaborative effort, we characterized 10 gcGBMs by chromosome studies, single nucleotide polymorphism microarray analysis, and targeted next-generation sequencing. These tumors were subsequently compared to the genomic and epigenomic profile of glioblastomas described in The Cancer Genome Atlas (TCGA) dataset. RESULTS: Our analysis identified a specific pattern of genome-wide massive loss of heterozygosity (LOH) driven by near haploidization in a subset of glioblastomas with giant cell histology. We compared the genomic signature of these tumors against that of all glioblastomas in the TCGA dataset (n = 367) and confirmed that our cohort of gcGBMs demonstrated a significantly different genomic profile. Integrated genomic and histologic review of the TCGA cohort identified 3 additional gcGBMs with a near haploid genomic profile. CONCLUSIONS: Massive LOH driven by haploidization represents a defining molecular hallmark of a subtype of gcGBM. This unusual mechanism of tumorigenesis provides a diagnostic genomic hallmark to evaluate in future cases, may explain reported differences in survival, and suggests new therapeutic vulnerabilities.

CNTN6 copy number variations: Uncertain clinical significance in individuals with neurodevelopmental disorders.

Copy number variations (CNVs) of the CNTN6 gene - a member of the contactin gene superfamily - have been previously proposed to have an association with neurodevelopmental and autism spectrum disorders. However, no functional evidence has been provided to date and phenotypically normal and mildly affected carriers complicate the interpretation of this aberration. In view of conflicting reports on the pathogenicity of CNVs involving CNTN6 and association with different phenotypes, we, independently, evaluated clinical features of nineteen patients with detected CNV of CNTN6 as part of their clinical microarray analysis at Children's Mercy and Nationwide Children's Hospitals for the period of 2008-2015. The clinical presentations of these patients were variable making it difficult to establish genotype-phenotype correlations. CNVs were inherited in six patients. For thirteen patients, inheritance pattern was not established due to unavailability of parental samples for testing. In three cases CNV was inherited from a healthy parent and in three cases from a parent with neurodevelopmental symptoms. Of the nineteen patients, four had a separate genetic abberation in addition to CNV of the CNTN6 that could independently explain their respective phenotypes. Separately, CNTN6 sequencing was performed on an autism spectrum disorder (ASD) research cohort of 94 children from 80 unrelated families. We found no difference in frequency of rare coding variants between the cohort of patients and controls. We conclude that CNVs involving CNTN6 alone seem to be most likely a neutral variant or a possible modifier rather than a disease-causing variant. Patients with CNVs encompassing CNTN6 could benefit from additional genetic testing since a clinical diagnosis due to a CNV of CNTN6 alone is still questionable.

Occurrence and characterization of medulloblastoma in a patient with Curry-Jones syndrome.

Medulloblastoma in a Patient with Curry-Jones Syndrome with a mosaic variant, c.1234C > T (p.Leu412Phe), in SMO.

Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC).

The detection of acquired copy-number abnormalities (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) in neoplastic disorders by chromosomal microarray analysis (CMA) has significantly increased over the past few years with respect to both the number of laboratories utilizing this technology and the broader number of tumor types being assayed. This highlights the importance of standardizing the interpretation and reporting of acquired variants among laboratories. To address this need, a clinical laboratory-focused workgroup was established to draft recommendations for the interpretation and reporting of acquired CNAs and CN-LOH in neoplastic disorders. This project is a collaboration between the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). The recommendations put forth by the workgroup are based on literature review, empirical data, and expert consensus of the workgroup members. A four-tier evidence-based categorization system for acquired CNAs and CN-LOH was developed, which is based on the level of available evidence regarding their diagnostic, prognostic, and therapeutic relevance: tier 1, variants with strong clinical significance; tier 2, variants with some clinical significance; tier 3, clonal variants with no documented neoplastic disease association; and tier 4, benign or likely benign variants. These recommendations also provide a list of standardized definitions of terms used in the reporting of CMA findings, as well as a framework for the clinical reporting of acquired CNAs and CN-LOH, and recommendations for how to deal with suspected clinically significant germline variants.

Neuroblastoma in Adolescents and Children Older than 10 Years: Unusual Clinicopathologic and Biologic Features.

Neuroblastoma (NB) in children older than 10 years is rare. We reviewed our archives for patients with NB aged 10 to 18 years and summarized their clinicopathologic/genetic records. Of 96 patients, 4 patients were identified in this age group. Four tumors were abdominal; 1 patient had 2 tumors at diagnosis, one of which was presacral. Tumor sizes ranged from 3 to 20 cm. All tumors were high risk at clinical stages 3 and 4, with metastasis to bone marrow and other areas. Four tumors were poorly differentiated with unfavorable histology and one patient with bilateral adrenal disease had an intermixed ganglioneuroblastoma on one side. Another tumor exhibited pheochromocytoma-like morphology. MYCN amplification was present in bone marrow metastasis in one case. Complex chromosomal gains and 19p deletions were common. Exome sequencing revealed ALK variants in 2 cases and previously unreported MAGI2, RUNX1, and MLL mutations. All patients received standard chemotherapy and 2 patients received ALK-targeted trial therapy. Three patients died of disease, ranging 18 to 23 months after diagnosis. One patient has active disease and is receiving trial therapy. In conclusion, NB in children older than 10 years may exhibit unusual clinicopathologic and genetic features with large tumors, bilateral adrenal disease, rare morphologic features, complex DNA microarray findings and novel mutations. Patients often have grim prognoses despite genomic profiling-guided targeted therapy.

Maintaining a methods database to optimize solid tumor tissue culture: Review of a 15-year database from a single institution.

Chromosome analysis of solid tumors provides valuable information for diagnosis and patient management, yet successfully culturing solid tumors can be challenging. The Children's Mercy (CM) Cytogenetics laboratory has compiled a database of 1371 non-lymphoma solid tumors cultured since 2002. Analysis of the tumor culture data found a culture success rate of 91.6%. Abnormal karyotypes were identified in 47.0% of these tumors. A quality improvement project reviewed the database for methods, cell culture success, yield of clonally abnormal karyotypes, culture failure, tumor diagnostic category, and other. This review revealed processes that could be optimized with minor changes to methods in a subset of tumors. Three tumor/method pair examples are provided including adrenal cortical carcinomas (ACCs), choroid plexus tumors (CPTs), and neuroblastoma. The successful culture of tumors as defined by capture of clonally abnormal cells is dependent upon several factors including culture medium, monolayer versus suspension culture, length of time in culture, method of disaggregation and other. The database serves as a quality assurance tool that enables continuous improvement in culture success rate and abnormal yield. It is also an educational resource for laboratory technologists, residents and fellows. Using the database to track methods and results ensures consistency in routine tumor processing, facilitates oversight to optimize methods for quality, and improves results for patient care.

Juvenile myelomonocytic leukemia with t(3;5)(q25;q35), Auer rods and marked myelodysplasia.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive childhood leukemia characterized by an excess proliferation of cells of granulocytic and monocytic lineages. The WHO classifies JMML with the myelodysplastic/myeloproliferative neoplasms. Myelodysplasia in JMML is usually minimal to mild. Auer rods have never been reported in JMML. We present a 2-year-old boy with splenomegaly, leukocytosis, thrombocytopenia, anemia, and excess myeloblasts with easily seen Auer rods, and marked dysgranulopoiesis and dyserythropoiesis. Conventional cytogenetic analysis showed a sole abnormality of t(3;5)(q25;q35). Microarray analysis showed a terminal 21 Mb region of copy-neutral loss of heterozygosity on 19q. Disease-related somatic NRAS mutation was detected. This case represents an unusual JMML with Auer rods and marked myelodysplasia. These unusual histopathologic features may be related to the t(3;5)(q25;q35). A t(3;5) with variable breakpoints has been reported in a small proportion of acute myeloid leukemias and myelodysplastic syndromes. To our knowledge, this is the first JMML case reported with this translocation.

Section E6.1-6.4 of the ACMG technical standards and guidelines: chromosome studies of neoplastic blood and bone marrow-acquired chromosomal abnormalities.

DISCLAIMER: These American College of Medical Genetics and Genomics standards and guidelines are developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these standards and guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Cytogenetic analyses of hematological neoplasms are performed to detect and characterize clonal chromosomal abnormalities that have important diagnostic, prognostic, and therapeutic implications. At the time of diagnosis, cytogenetic abnormalities assist in the diagnosis of such disorders and can provide important prognostic information. At the time of relapse, cytogenetic analysis can be used to confirm recurrence of the original neoplasm, detect clonal disease evolution, or uncover a new unrelated neoplastic process. This section deals specifically with the standards and guidelines applicable to chromosome studies of neoplastic blood and bone marrow-acquired chromosomal abnormalities. This updated Section E6.1-6.4 has been incorporated into and supersedes the previous Section E6 in Section E: Clinical Cytogenetics of the 2009 Edition (Revised 01/2010), American College of Medical Genetics and Genomics Standards and Guidelines for Clinical Genetics Laboratories.Genet Med 18 6, 635-642.

Section E6.5-6.8 of the ACMG technical standards and guidelines: chromosome studies of lymph node and solid tumor-acquired chromosomal abnormalities.

DISCLAIMER: These ACMG standards and guidelines are developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these standards and guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Cytogenetic analysis of tumor tissue is performed to detect and characterize chromosomal aberrations to aid histopathological and clinical diagnosis and patient management. At the time of diagnosis, known recurrent clonal aberrations may facilitate histopathological diagnosis and subtyping of the tumor. This information may contribute to clinical therapeutic decisions. However, even when tumors have a known recurrent clonal aberration, each tumor is genetically unique and probably heterogeneous. It is important to discover as much about the genetics of a tumor at diagnosis as is possible with the methods available for study of the tumor material. The information gathered at initial study will inform follow-up studies, whether for residual disease detection, determination of relapse and clonal evolution, or identifying a new disease clone.This updated Section E6.5-6.8 has been incorporated into and supersedes the previous Sections E6.4 and E6.5 in Section E: Clinical Cytogenetics of the 2009 Edition (Revised 01/2010), American College of Medical Genetics and Genomics Standards and Guidelines for Clinical Genetics Laboratories. This section deals specifically with the standards and guidelines applicable to lymph node and solid tumor chromosome analysis.Genet Med 18 6, 643-648.

Clinical detection of deletion structural variants in whole-genome sequences.

Optimal management of acutely ill infants with monogenetic diseases requires rapid identification of causative haplotypes. Whole-genome sequencing (WGS) has been shown to identify pathogenic nucleotide variants in such infants. Deletion structural variants (DSVs, >50 nt) are implicated in many genetic diseases, and tools have been designed to identify DSVs using short-read WGS. Optimisation and integration of these tools into a WGS pipeline could improve diagnostic sensitivity and specificity of WGS. In addition, it may improve turnaround time when compared with current CNV assays, enhancing utility in acute settings. Here we describe DSV detection methods for use in WGS for rapid diagnosis in acutely ill infants: SKALD (Screening Konsensus and Annotation of Large Deletions) combines calls from two tools (Breakdancer and GenomeStrip) with calibrated filters and clinical interpretation rules. In four WGS runs, the average analytic precision (positive predictive value) of SKALD was 78%, and recall (sensitivity) was 27%, when compared with validated reference DSV calls. When retrospectively applied to a cohort of 36 families with acutely ill infants SKALD identified causative DSVs in two. The first was heterozygous deletion of exons 1-3 of MMP21 in trans with a heterozygous frame-shift deletion in two siblings with transposition of the great arteries and heterotaxy. In a newborn female with dysmorphic features, ventricular septal defect and persistent pulmonary hypertension, SKALD identified the breakpoints of a heterozygous, de novo 1p36.32p36.13 deletion. In summary, consensus DSV calling, implemented in an 8-h computational pipeline with parameterised filtering, has the potential to increase the diagnostic yield of WGS in acutely ill neonates and discover novel disease genes.

MMP21 is mutated in human heterotaxy and is required for normal left-right asymmetry in vertebrates.

Heterotaxy results from a failure to establish normal left-right asymmetry early in embryonic development. By whole-exome sequencing, whole-genome sequencing and high-throughput cohort resequencing, we identified recessive mutations in MMP21 (encoding matrix metallopeptidase 21) in nine index cases with heterotaxy. In addition, Mmp21-mutant mice and mmp21-morphant zebrafish displayed heterotaxy and abnormal cardiac looping, respectively, suggesting a new role for extracellular matrix remodeling in the establishment of laterality in vertebrates.

Spitzoid melanoma in a child with Li-Fraumeni syndrome.

Spitzoid melanoma of childhood is a rare malignancy. The histological features are at the upper end of a range encompassing Spitz nevus and atypical Spitz tumor, the unifying features including large oval, fusiform or polygonal melanocytes with abundant homogeneous-appearing cytoplasma and large vesicular nuclei. The presence of a "bottom-heavy" pattern, strikingly enlarged nuclei and nucleoli in both the upper and lower portions of the lesion, and deep mitotic figures are among the findings that distinguish most of the Spitzoid melanomas from Spitz nevi and atypical Spitz tumors. There are no syndromic associations reported for this malignancy. We report the occurrence of choroid plexus carcinoma, Spitzoid melanoma, and myelodysplasia in a child who was found to carry a germline mutation for TP53. While choroid plexus carcinoma and myelodysplasia have relatively frequently been described, melanomas have been very rarely described in Li-Fraumeni syndrome. The association of Spitzoid melanoma with Li-Fraumeni syndrome, especially in a pediatric patient, has not been reported before.