Fluorescence in situ hybridization (FISH) remains the gold-standard clinical assay to detect genetic abnormalities in multiple myeloma (MM). However, FISH panel design, use of conventional chromosome banding analysis and reporting practices have been reported to vary among laboratories. Therefore, standardization in FISH testing and reporting practices is needed to improve report clarity and avoid misinterpretation. The recommendations in this paper represent a consensus of our Cancer Genomics Consortium Plasma Cell Neoplasm Working Group, comprising a joint panel of cytogenetic laboratory directors and clinical investigators with expertise in the diagnosis, risk stratification, and treatment of multiple myeloma. Prior to developing these consensus recommendations, we performed a full literature review and conducted a survey of 102 oncologists to assess current variations and challenges in MM cytogenetic/FISH testing and reporting. Our guidelines establish best practices for the optimization of FISH panel selection, and recommendations for standardized reporting of cytogenetic results to align with the 2025 International Myeloma Society (IMS)/International Myeloma Working Group (IMWG) Updated Risk Stratification.

Purpose Multiple myeloma (MM) remains an incurable plasma cell malignancy with recurrent primary and secondary cytogenetic abnormalities having prognostic and therapeutic implications. Fluorescence in situ hybridization (FISH) is the gold-standard assay to detect these genetic abnormalities. However, FISH testing for MM is heterogeneous among clinical laboratories, with differences in plasma cell isolation, FISH panel design, and reporting practices. Methods The CGC Plasma Cell Neoplasm workgroup conducted a survey targeting the international MM clinician community on utilization of FISH and result reporting/interpretation. Results There were 102 survey responses representing 14 countries. Most (74%) MM clinicians utilize their own in-house FISH testing service with 81% reporting plasma cell enrichment was performed by their lab. 90% of respondents desired FISH at diagnosis, 72% during disease progression and 40% for treatment/response assessment. The most-requested FISH probes included: TP53 (99%), t(4;14) (92%), 1q gain/amplification (91%), t(14;16) (90%), t(11;14) (85%), t(14;20) (76%), 1p deletion (67%), while FISH for ploidy status, deletion 13q/-13, t(6;14), MYC rearrangement, and other rare IG rearrangements were ranked lower in importance (10-50%). About 40% of respondents were dissatisfied with clarity, summary, and interpretation of FISH reports. When challenged to interpret a FISH report, only 2% of responders interpreted results correctly and the majority were either unsure or misinterpreted the report. Conclusion Our study showed that significant improvements are needed by clinical lab directors in MM FISH report clarity to benefit both the clinician and patient. We propose standardization of best MM FISH practices and reporting.

Clinical cytogenomic studies of solid tumor samples are critical to the diagnosis, prognostication, and treatment selection for cancer patients. An overview of current cytogenomic techniques for solid tumor analysis is provided, including standards for sample preparation, clinical and technical considerations, and documentation of results. With the evolving technologies and their application in solid tumor analysis, these standards now include sequencing technology and optical genome mapping, in addition to the conventional cytogenomic methods, such as G-banded chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray analysis. This updated Section E6.7-6.12 supersedes the previous Section E6.5-6.8 in Section E: Clinical Cytogenetics of the American College of Medical Genetics and Genomics Standards for Clinical Genetics Laboratories.

Genome-wide uniparental disomy (GWUPD), or uniparental diploidy, is a condition in which the entire genome is inherited from a single parent. It is only compatible with life when chimeric or mosaic with a normal cell line. Previous studies have suggested different mechanisms for chimeric or mosaic GWUPD formation involving meiotic and/or mitotic events. To date, about 32 live-born individuals with chimeric or mosaic GWUPD have been reported in the literature. The majority are females that showed mosaic GW paternal UPD (GWpatUPD) based on isodisomies.

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 thorn 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.& COPY; 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.

The Sarcoma Working Group of the Cancer Genomics Consortium (CGC) aims to understand the current state of diagnostic testing for pediatric sarcomas and the challenges faced in the field. We conducted a survey of CGC general constituents and American Cytogenetics Forum members ([email protected]) in April/May 2022. In total, 46 responses were received, predominantly from university/academic/teaching hospitals (n=45) in the United States (n=41) and Canada (n=5). Respondents reported diverse roles including laboratory directors (n=25), pathologists (n=15), oncologists (n=3), and laboratory technologists (n=3). While most participants reported having in-house FISH (n=40) and/or conventional karyotyping (n=28) assays available for pediatric sarcomas, less than 40% of laboratories offer microarray and/or comprehensive NGS testing clinically. Detailed information on methodology and reported variant types will be discussed. When asked about genetic testing conundrums faced in their practice, the most common response was the scarcity of testing algorithm guidelines (n=12). Additional recurring challenges included: lack of support for new test development and issues with sendout testing (n=6), insufficient knowledge to interpret and act on results (n=4) or when to perform germline testing (n=3), limited tissue for comprehensive testing (n=3), and insurance/reimbursement complications (n=3). To address gaps in guidance for testing, the Workgroup is preparing a manuscript that will report survey results, provide practical scenario-based examples informed by the survey results and supported by literature evidence, and discuss testing options in resource-limited settings for the diagnostic workup of pediatric sarcomas. Areas of focus include but are not limited to NTRK-rearranged tumors and relevant germline testing in pediatric sarcomas.

These standards introduced a categorization system for the interpretation and reporting of acquired CNAs and CN-LOH in neoplastic disorders (PMID: 31138931). Three years after their publication, the workgroup developed a survey to gauge implementation of the standards in clinical laboratories. Part 1 of the survey was focused on clinical implementation data and part 2 on laboratory practices. The survey was distributed through CGC, and 32 responses from 4 continents were received. 75% of participants reported that they either fully or partially implemented the standards in their laboratories. All participants who implemented the standards introduced no or minor modifications. The biggest challenges to implementation included the perceived ambiguity in tier definitions, increased time/labor for interpretation of CNAs and CN-LOH following the standards, and limitations of analytic software. 56% of participants reported satisfaction with the 4-tier classification system, 31% were neutral, 10% had no opinion, and 3% were not satisfied. The top three recommended changes involved clarification of the tiers, including 1) provide more examples of different tiers, 2) provide more detailed description of criteria for each tier, and 3) provide additional education on how to apply the tier classification system. Detailed analysis of the survey data will be presented, including responses regarding the utility of the tier examples, definition of terms, and recommended report structure, as well as laboratory practices. The large amount of data collected on the survey will guide the future revision of the standards aimed at standardization and continued improvement of laboratory practices.

ETS-related gene ( ERG ) amplification, observed in 4-6% of acute myeloid leukemia (AML), is associated with unfavorable prognosis. To determine coincident effects of additional genomic abnormalities in AML with ERG amplification (ERGamp), we examined 11 ERGamp cases of 205 newly diagnosed AML using chromosomal microarray analysis and next generation sequencing. ERGamp cases demonstrated a dis-tinct pattern of high genetic complexity: loss of 5q, chromothripsis and TP53 loss of function variants. Remarkably, allelic TP53 loss or loss of heterozygosity (LOH) co-occurring with TP53 inactivating mu-tation dramatically effected ERGamp tumor patient outcome. In the presence of homozygous TP53 loss of function, ERGamp patients demonstrated no response to induction chemotherapy with median overall survival (OS) of 3.8 months ( N = 9). Two patients with heterozygous loss of TP53 function underwent alloSCT without evidence of relapse at one year. Similarly, a validation TCGA cohort, 6 of the 8 ERGamp cases with TP53 loss of function demonstrated median OS of 2.5 months. This suggests that with TP53 mutant ERGamp AML, successive loss of the second TP53 allele, typically by 17p deletion or LOH identi-fies a specific high-risk subtype of AML patients who are resistant to standard induction chemotherapy and need novel approaches to avert the very poor prognosis.(c) 2023 Elsevier Inc. All rights reserved.

We report a case of a somatic overgrowth syndrome diagnosed at forensic autopsy with the aid of next generation sequencing as Proteus syndrome. Somatic overgrowth syndromes result from spontaneous somatic mutations that arise early in development and display a mosaic pattern of expression in patient tissues. Due to the temporal and anatomic heterogeneity of these syndromes, phenotypes vary widely, resulting in clinical overlap. Furthermore, the variable ratio of mutated to nonmutated cells in patient tissue can result in low-level mutations that could be missed using Sanger sequencing. Due to these factors, recent literature points to next generation sequencing (NGS) as an adjunct to diagnosis of these rare entities. A male in his fourth decade of life presented to our forensic autopsy service with physical features suggestive of a somatic overgrowth syndrome. Due to the paucity of clinical information accompanying the individual, a definitive diagnosis based on physical characteristics, alone, was not possible. Next generation sequencing of affected formalin-fixed and paraffin-embedded brain tissue confirmed the presence of the variant in AKT1 (c.49G>A, p.Glu17Lys, in 14.13% of reads) found in Proteus syndrome. To our knowledge, this is the first report of the mosaic variant of AKT1 detected in brain tissue and the first reported case of a postmortem diagnosis of Proteus syndrome with the aid of NGS. We conclude that NGS can be used as an adjunctive method to support a specific diagnosis among the somatic overgrowth syndromes postmortem in the absence of sufficient clinical history.

Chromosomal microarray analysis (CMA) is instrumental for identification of recurrent focal deletions in patients with B cell acute lymphoblastic leukemia (B-ALL). These microdeletions include genes involved in B cell differentiation, cell cycle control and transcriptional regulation often with prognostic or therapeutic significance. Recently, a 13q12.2 microdeletion with breakpoints upstream of FLT3 and within intronic regions of the PAN3 gene was reported. Functionally, this microdeletion resulted in enhancer hijacking and constitutive activation of FLT3 in pediatric B-ALL. Seven institutions performed internal database searches to identify similar deletions. In total, 10 patients were identified, all with B-ALL, confirming the specificity for this diagnosis. This cohort included seven pediatric cases (3 male, 4 female; ages 2-14 yo, mean 5) and 3 adult cases (1 male, 2 female; ages 22, 39, 42). Subtypes included two high hyperdiploid, one hypodiploid, one iAMP21, one P2RY8::CLRF, one unbalanced rearrangement involving TFE3, one PAX5::PML, three B-ALL NOS.? Deletions ranged in size from?6.7 kb ? 136 kb (mean 76 kb); each with proximal breakpoints between FLT3 and PAN3 and 9/10 with distal breakpoints within intron 5 of the PAN3 gene. Our study is the first to report the microdeletion in adults, and unlike the discovery cohorts, our patients showed no sex bias. Our data does not confirm a strong association of the deletion with high hyperdiploidy, since 8/10 had other B-ALL subtypes. Constitutive activation of FLT3 may have significant clinical implications for prognosis and treatment; thus, detection of 13q12.2 microdeletion at diagnosis or disease relapse is paramount. Chromosomal microarray analysis (CMA) is instrumental for identification of recurrent focal deletions in patients with B cell acute lymphoblastic leukemia (B-ALL). These microdeletions include genes involved in B cell differentiation, cell cycle control and transcriptional regulation often with prognostic or therapeutic significance. Recently, a 13q12.2 microdeletion with breakpoints upstream of FLT3 and within intronic regions of the PAN3 gene was reported. Functionally, this microdeletion resulted in enhancer hijacking and constitutive activation of FLT3 in pediatric B-ALL. Seven institutions performed internal database searches to identify similar deletions. In total, 10 patients were identified, all with B-ALL, confirming the specificity for this diagnosis. This cohort included seven pediatric cases (3 male, 4 female; ages 2-14 yo, mean 5) and 3 adult cases (1 male, 2 female; ages 22, 39, 42). Subtypes included two high hyperdiploid, one hypodiploid, one iAMP21, one P2RY8::CLRF, one unbalanced rearrangement involving TFE3, one PAX5::PML, three B-ALL NOS.? Deletions ranged in size from?6.7 kb ? 136 kb (mean 76 kb); each with proximal breakpoints between FLT3 and PAN3 and 9/10 with distal breakpoints within intron 5 of the PAN3 gene. Our study is the first to report the microdeletion in adults, and unlike the discovery cohorts, our patients showed no sex bias. Our data does not confirm a strong association of the deletion with high hyperdiploidy, since 8/10 had other B-ALL subtypes. Constitutive activation of FLT3 may have significant clinical implications for prognosis and treatment; thus, detection of 13q12.2 microdeletion at diagnosis or disease relapse is paramount.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofibromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodeficient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identified in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identified mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9. This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs.

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.

Giant cell glioblastoma (GCG) is a rare histologic variant of glioblastoma that is characterized by numerous bizarre, multinucleate giant cells and abundant reticulin deposition. GCG occurs in younger patients (mean age, 44 years) and generally has a better prognosis than other variants of IDH-wildtype glioblastoma. Although prior studies have identified frequent TP53 alterations in these tumors, the genomics of GCG remain poorly characterized. Herein, we describe an inter-institutional analysis of seven patients ranging from age 9 to 68 of variable race and gender diagnosed with GCG. Across 100% (7/7) of specimens in our cohort, chromosomal microarray analysis and targeted next generation sequencing consistently identified near haploidization / massive loss of heterozygosity (LOH). Remarkably, the genomic profile of these tumors showed LOH of chromosomes 3, 4, 6, 8, 13, 14, 15, 17, 22 in all cases (7/7), and LOH of chromosomes 1, 2, 5, 9, 10, 11, 18, 19 in 6/7 cases. Notably, chromosome 7 always retained heterozygosity. We propose that this genomic profile represents a distinct subtype of GCG. These findings provide evidence of an unusual mechanism of tumorigenesis and could have the potential to reveal new tumor vulnerability.