The New Era of Cancer Cytogenetics and Cytogenomics.

The promises of the cancer genome sequencing project, combined with various -omics technologies, have raised questions about the importance of cancer cytogenetic analyses. It is suggested that DNA sequencing provides high resolution, speed, and automation, potentially replacing cytogenetic testing. We disagree with this reductionist prediction. On the contrary, various sequencing projects have unexpectedly challenged gene theory and highlighted the importance of the genome or karyotype in organizing gene network interactions. Consequently, profiling the karyotype can be more meaningful than solely profiling gene mutations, especially in cancer where karyotype alterations mediate cellular macroevolution dominance. In this chapter, recent studies that illustrate the ultimate importance of karyotype in cancer genomics and evolution are briefly reviewed. In particular, the long-ignored non-clonal chromosome aberrations or NCCAs are linked to genome or chromosome instability, genome chaos is linked to genome reorganization under cellular crisis, and the two-phased cancer evolution reconciles the relationship between genome alteration-mediated punctuated macroevolution and gene mutation-mediated stepwise microevolution. By further synthesizing, the concept of karyotype coding is discussed in the context of information management. Altogether, we call for a new era of cancer cytogenetics and cytogenomics, where an array of technical frontiers can be explored further, which is crucial for both basic research and clinical implications in the cancer field.

The Digital World of Cytogenetic and Cytogenomic Web Resources.

The dynamic growth of technological capabilities at the cellular and molecular level has led to a rapid increase in the amount of data on the genes and genomes of organisms. In order to store, access, compare, validate, classify, and understand the massive data generated by different researchers, and to promote effective communication among research communities, various genome and cytogenetic online databases have been established. These data platforms/resources are essential not only for computational analyses and theoretical syntheses but also for helping researchers select future research topics and prioritize molecular targets. Furthermore, they are valuable for identifying shared recurrent genomic patterns related to human diseases and for avoiding unnecessary duplications among different researchers. The website interface, menu, graphics, animations, text layout, and data from databases are displayed by a front end on the screen of a monitor or smartphone. A database front-end refers to the user interface or application that enables accessing tabular, structured, or raw data stored in the database. The Internet makes it possible to reach a greater number of users around the world and gives them quick access to information stored in databases. The number of ways of presenting this data by front-ends increases as well. This requires unifying the ways of operating and presenting information by front-ends and ensuring contextual switching between front-ends of different databases. This chapter aims to present selected cytogenetic and cytogenomic Internet resources in terms of obtaining the needed information and to indicate how to increase the efficiency of access to stored information. Through a brief introduction of these databases and by providing examples of their usage in cytogenetic analyses, we aim to bridge the gap between cytogenetics and molecular genomics by encouraging their utilization.

Cancer cytogenetics in a genomics world: Wedding the old with the new.

Since the discovery of the Philadelphia chromosome in 1960, cytogenetic studies have been instrumental in detecting chromosomal abnormalities that can inform cancer diagnosis, treatment, and risk assessment efforts. The initial expansion of cancer cytogenetics was with fluorescence in situ hybridization (FISH) to assess submicroscopic alterations in dividing or non-dividing cells and has grown into the incorporation of chromosomal microarrays (CMA), and next generation sequencing (NGS). These molecular technologies add additional dimensions to the genomic assessment of cancers by uncovering cytogenetically invisible molecular markers. Rapid technological and bioinformatic advances in NGS are so promising that the idea of performing whole genome sequencing as part of routine patient care may soon become economically and logistically feasible. However, for now cytogenetic studies continue to play a major role in the diagnostic testing and subsequent assessments in leukemia with other genomic studies serving as complementary testing options for detection of actionable genomic abnormalities. In this review, we discuss the role of conventional cytogenetics (karyotyping, chromosome analysis) and FISH studies in hematological malignancies, highlighting the continued clinical utility of these techniques, the subtleties and complexities that are relevant to treating physicians and the unique strengths of cytogenetics that cannot yet be paralleled by the current high-throughput molecular technologies. Additionally, we describe how CMA, optical genome mapping (OGM), and NGS detect abnormalities that were beyond the capacity of cytogenetic studies and how an integrated approach (broad molecular testing) can contribute to the detection of actionable targets and variants in malignancies. Finally, we discuss advances in the field of genomic testing that are bridging the advantages of individual (single) cell based cytogenetic testing and broad genomic testing.

Clinical Cytogenetics: Current Practices and Beyond.

BACKGROUND: Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics. CONTENT: In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements. SUMMARY: Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity.

Complementary Tumor Diagnosis by Single Cell-Based Cytogenetics Using Multi-marker Fluorescence In Situ Hybridization (mFISH).

Multi-color (or multi-marker) fluorescence in situ hybridization (mFISH) is a well-established, valuable, complementary tool for prenatal and pathological (tumor) diagnosis. A variety of chromosomal abnormalities, such as partial or total chromosomal gains, losses, inversions, or translocations, which are considered to cause genetic syndromes, can relatively easily be detected on a cell-by-cell basis. Individual cells either in suspension (e.g., in the form of a cytological specimen derived from body fluids) or within a tissue (e.g., a solid tumor specimen or biopsy) can be quantitatively evaluated with respect to the chromosomal hybridization markers of interest (e.g., a gene or centromeric region) and with due consideration of cellular heterogeneity. FISH is helpful or even essential for the (sub-)classification, stratification, and unambiguous diagnosis of a number of malignant diseases and contributes to treatment decision in many cases. Here, the diagnostic power and limitations of typical FISH and mFISH approaches (except chromosome painting and RNA hybridization) are discussed, with special emphasis on tumor and single-cell diagnostics. Well-established and novel FISH protocols, the latter addressed to accelerate and flexibilize the preparation and hybridization of formalin-fixed and paraffin-embedded tissues, are provided. Moreover, guidelines and molecular aspects important for data interpretation are discussed. Finally, sophisticated multiplexed approaches and those that analyze very rare single-cell events, which are not yet implemented in diagnostic procedures, will be touched upon. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: (m)FISH applied to formaldehyde-fixed paraffin-embedded tissues Basic Protocol 2: (m)FISH applied to cytological specimens.

Plant Cytogenetics: From Chromosomes to Cytogenomics.

Chromosomes have been studied since the late nineteenth century in the disciplines of cytology and cytogenetics. Analyzing their numbers, features, and dynamics has been tightly linked to the technical development of preparation methods, microscopes, and chemicals to stain them, with latest continuing developments described in this volume. At the end of the twentieth and beginning of the twenty-first centuries, DNA technology, genome sequencing, and bioinformatics have revolutionized how we see, use, and analyze chromosomes. The advent of in situ hybridization has shaped our understanding of genome organization and behavior by linking molecular sequence information with the physical location along chromosomes and genomes. Microscopy is the best technique to accurately determine chromosome number. Many features of chromosomes in interphase nuclei or pairing and disjunction at meiosis, involving physical movement of chromosomes, can only be studied by microscopy. In situ hybridization is the method of choice to characterize the abundance and chromosomal distribution of repetitive sequences that make up the majority of most plant genomes. These most variable components of a genome are found to be species- and occasionally chromosome-specific and give information about evolution and phylogeny. Multicolor fluorescence hybridization and large pools of BAC or synthetic probes can paint chromosomes and we can follow them through evolution involving hybridization, polyploidization, and rearrangements, important at a time when structural variations in the genome are being increasingly recognized. This volume discusses many of the most recent developments in the field of plant cytogenetics and gives carefully compiled protocols and useful resources.

Tools for Drawing Informative Idiograms.

Each species has a typical karyotype, which represents the phenotypic appearance of the somatic chromosomes including number, size, and morphology. An idiogram is a diagrammatic representation of the chromosomes showing their relative size, homologous groups, and different cytogenetic landmarks. Chromosomal analysis of cytological preparations is an essential component of many investigations, which involves the calculation of karyotypic parameters and the generation of idiograms. Although various tools are available for karyotype analysis, here we demonstrate karyotype analysis using our recently developed tool named KaryoMeasure. KaryoMeasure is a semi-automated free and user-friendly karyotype analysis software that facilitates data collection from different digital images of metaphase chromosome spreads and calculates a wide variety of chromosomal and karyotypic parameters along with the related standard errors. KaryoMeasure draws idiograms of both diploid and allopolyploid species into a vector-based SVG or PDF image file.

Challenges and Opportunities for Clinical Cytogenetics in the 21st Century.

The powerful utilities of current DNA sequencing technology question the value of developing clinical cytogenetics any further. By briefly reviewing the historical and current challenges of cytogenetics, the new conceptual and technological platform of the 21st century clinical cytogenetics is presented. Particularly, the genome architecture theory (GAT) has been used as a new framework to emphasize the importance of clinical cytogenetics in the genomic era, as karyotype dynamics play a central role in information-based genomics and genome-based macroevolution. Furthermore, many diseases can be linked to elevated levels of genomic variations within a given environment. With karyotype coding in mind, new opportunities for clinical cytogenetics are discussed to integrate genomics back into cytogenetics, as karyotypic context represents a new type of genomic information that organizes gene interactions. The proposed research frontiers include: 1. focusing on karyotypic heterogeneity (e.g., classifying non-clonal chromosome aberrations (NCCAs), studying mosaicism, heteromorphism, and nuclear architecture alteration-mediated diseases), 2. monitoring the process of somatic evolution by characterizing genome instability and illustrating the relationship between stress, karyotype dynamics, and diseases, and 3. developing methods to integrate genomic data and cytogenomics. We hope that these perspectives can trigger further discussion beyond traditional chromosomal analyses. Future clinical cytogenetics should profile chromosome instability-mediated somatic evolution, as well as the degree of non-clonal chromosomal aberrations that monitor the genomic system's stress response. Using this platform, many common and complex disease conditions, including the aging process, can be effectively and tangibly monitored for health benefits.

Evaluation of optical genome mapping for detecting chromosomal translocation in clinical cytogenetics.

BACKGROUND: Balanced reciprocal translocation is one of the most common chromosomal abnormalities in humans that may lead to infertility, recurrent pregnancy loss, or having children with physical or mental abnormalities. Karyotyping and FISH are traditional detection approaches with a low resolution. Bionano optical genome mapping (OGM) developed in recent years can be used to analyze chromosomal abnormalities at a higher resolution, providing the possibility of more in-depth analyses of balanced chromosome translocations. METHODS: To evaluate the feasibility of OGM to detect chromosome balanced translocations, 10 genetic outpatients were collected and detected simultaneously by karyotype analysis, FISH, CNV-seq, and Bionano OGM in this study. RESULTS: The results showed that the karyotypes of the patients were detected by karyotype analysis, FISH, and Bionano OGM, but one patient with karyotype t(Y,19) was not correctly detected by OGM. There were not find any chromosome abnormality by CNV-seq. More importantly, OGM allowed the location of the mutation to the gene level, which is important for aiding diagnoses, compared to karyotype analysis, and FISH. CONCLUSIONS: This study shows that OGM can be a high adjunctive diagnostic method for detecting balanced chromosome translocations, but the accuracy and precision of OGM detecting mutations need to be gradually improved in telomere and centromere regions.

Progress in Methods for Copy Number Variation Profiling.

Copy number variations (CNVs) are the predominant class of structural genomic variations involved in the processes of evolutionary adaptation, genomic disorders, and disease progression. Compared with single-nucleotide variants, there have been challenges associated with the detection of CNVs owing to their diverse sizes. However, the field has seen significant progress in the past 20-30 years. This has been made possible due to the rapid development of molecular diagnostic methods which ensure a more detailed view of the genome structure, further complemented by recent advances in computational methods. Here, we review the major approaches that have been used to routinely detect CNVs, ranging from cytogenetics to the latest sequencing technologies, and then cover their specific features.

Plant Cytogenetics in the Micronuclei Investigation-The Past, Current Status, and Perspectives.

Cytogenetic approaches play an essential role as a quick evaluation of the first genetic effects after mutagenic treatment. Although labor-intensive and time-consuming, they are essential for the analyses of cytotoxic and genotoxic effects in mutagenesis and environmental monitoring. Over the years, conventional cytogenetic analyses were a part of routine laboratory testing in plant genotoxicity. Among the methods that are used to study genotoxicity in plants, the micronucleus test particularly represents a significant force. Currently, cytogenetic techniques go beyond the simple detection of chromosome aberrations. The intensive development of molecular biology and the significantly improved microscopic visualization and evaluation methods constituted significant support to traditional cytogenetics. Over the past years, distinct approaches have allowed an understanding the mechanisms of formation, structure, and genetic activity of the micronuclei. Although there are many studies on this topic in humans and animals, knowledge in plants is significantly limited. This article provides a comprehensive overview of the current knowledge on micronuclei characteristics in plants. We pay particular attention to how the recent contemporary achievements have influenced the understanding of micronuclei in plant cells. Together with the current progress, we present the latest applications of the micronucleus test in mutagenesis and assess the state of the environment.

Molecular Cytogenetic and Y Copy Number Analysis of a Reciprocal ECAY-ECA13 Translocation in a Stallion with Complete Meiotic Arrest.

We present a detailed molecular cytogenetic analysis of a reciprocal translocation between horse (ECA) chromosomes Y and 13 in a Friesian stallion with complete meiotic arrest and azoospermia. We use dual-color fluorescence in situ hybridization with select ECAY and ECA13 markers and show that the translocation breakpoint in ECAY is in the multicopy region and in ECA13, at the centromere. One resulting derivative chromosome, Y;13p, comprises of ECAY heterochromatin (ETSTY7 array), a small single copy and partial Y multicopy region, and ECA13p. Another derivative chromosome 13q;Y comprises of ECA13q and most of the single copy ECAY, the pseudoautosomal region and a small part of the Y multicopy region. A copy number (CN) analysis of select ECAY multicopy genes shows that the Friesian stallion has significantly (p < 0.05) reduced CNs of TSPY, ETSTY1, and ETSTY5, suggesting that the translocation may not be completely balanced, and genetic material is lost. We discuss likely meiotic behavior of abnormal chromosomes and theorize about the possible effect of the aberration on Y regulation and the progression of meiosis. The study adds a unique case to equine clinical cytogenetics and contributes to understanding the role of the Y chromosome in male meiosis.

Cytogenetics from the Dock: A New Postmortem Protocol Makes It Possible to Obtain Cytogenetic Preparations from Marine Fish.

Cytogenetic studies in marine fish are scarce, and elemental cytogenetic information is available for not >2% of the species. Traditional cytogenetic methods require living individuals for their application, making the analysis of marine ichthyofauna very difficult. In this study, we present a detailed new protocol to obtain cytogenetic preparations from marine fish, through access to specimens in postmortem condition. The application of this protocol made it possible to access elemental cytogenetic information (diploid number) in six native species of the South Pacific Ocean, representative of five orders. In this way, we provide a new low-cost methodological tool for focused or large-scale cytogenetic analysis, both in economically important, native, or threatened species.

Cytogenetic Damage of Human Lymphocytes in Humanized Mice Exposed to Neutrons and X Rays 24 h After Exposure.

Detonation of an improvised nuclear device highlights the need to understand the risk of mixed radiation exposure as prompt radiation exposure could produce significant neutron and gamma exposures. Although the neutron component may be a relatively small percentage of the total absorbed dose, the large relative biological effectiveness (RBE) can induce larger biological DNA damage and cell killing. The objective of this study was to use a hematopoietically humanized mouse model to measure chromosomal DNA damage in human lymphocytes 24 h after in vivo exposure to neutrons (0.3 Gy) and X rays (1 Gy). The human dicentric and cytokinesis-block micronucleus assays were performed to measure chromosomal aberrations in human lymphocytes in vivo from the blood and spleen, respectively. The mBAND assay based on fluorescent in situ hybridization labeling was used to detect neutron-induced chromosome 1 inversions in the blood lymphocytes of the neutron-irradiated mice. Cytogenetics endpoints, dicentrics and micronuclei showed that there was no significant difference in yields between the 2 irradiation types at the doses tested, indicating that neutron-induced chromosomal DNA damage in vivo was more biologically effective (RBE ∼3.3) compared to X rays. The mBAND assay, which is considered a specific biomarker of high-LET neutron exposure, confirmed the presence of clustered DNA damage in the neutron-irradiated mice but not in the X-irradiated mice, 24 h after exposure.

Técnicas de citogenética y biología molecular para el diagnóstico y seguimiento de la leucemia promielocítica / Cytogenetic and molecular biology techniques for the diagnosis and monitoring of promyelocytic leukemia

Introducción: La leucemia promielocítica es un subtipo de leucemia mieloide aguda que se presenta frecuentemente con una coagulopatía potencialmente mortal, por lo que representa una emergencia médica. En la gran mayoría de los pacientes ocurre la t(15;17)(q24;q21) que genera el gen aberrante PML-RARA. Mediante diferentes técnicas de citogenética y de la biología molecular que detectan dichas aberraciones es posible diagnosticar la entidad de manera inequívoca y estudiar la enfermedad mínima residual. Objetivo: Describir, comparar y analizar las técnicas de citogenética y de la biología molecular que son útiles para el diagnóstico y el seguimiento del paciente con leucemia promielocítica. Así como señalar sus ventajas y limitaciones. Métodos: Se realizó revisión de la bibliografía científica de los últimos cinco años relacionada con el tema a través de PUBMED. Se realizó análisis y resumen de la información. Análisis y síntesis de la información: Se describen dos técnicas de citogenética y tres moleculares basadas en la aplicación de la reacción en cadena de la polimerasa. Se comparan y analizan sus ventajas y limitaciones. Conclusiones: Algunas de estas técnicas son útiles únicamente para el diagnóstico, mientras que otras, por su alta sensibilidad, se recomiendan para el seguimiento del paciente con leucemia promielocítica(AU)

Introduction: Promyelocytic leukemia (PML) is a subtype of acute myeloid leukemia that frequently presents with a potentially fatal coagulopathy, therefore it represents a medical emergency. In the vast majority of patients, the t (15; 17) (q24; q21) occurs, which generates the aberrant gene PML-RARA. Using different cytogenetic and molecular biology techniques that detect these aberrations, it is possible to unequivocally diagnose the entity and study minimal residual disease. Objective: To describe, compare and analyze cytogenetics and molecular biology techniques that are useful for diagnosis and follow-up of the patient with Promyelocytic leukemia. As well as pointing out its advantages and limitations. Methods: A review of the scientific bibliography of the last five years related to the subject was carried out through PUBMED. An analysis and summary of the information was made. Analysis and synthesis of the information: Two cytogenetic and three molecular techniques are described based on the application of the polymerase chain reaction. Its advantages and limitations are compared and analyzed. Conclusions: Some of these techniques are only useful for diagnosis, while others, due to their high sensitivity, are recommended for monitoring the patient with Promyelocytic leukemia(AU)

Refined pancreatobiliary UroVysion criteria and an approach for further optimization.

Pancreatobiliary strictures are a common source of false negatives for malignancy detection. UroVysion is more sensitive than any other method but remains underutilized because of conflicting sensitivities and specificities due to a lack of standardized cutoff criteria and confusion in interpreting results in the context of primary sclerosing cholangitis. We set out to determine the sensitivities and specificities of UroVysion, brushing cytology, forceps biopsies, and fine needle aspiration (FNAs) for pancreatobiliary stricture malignancy detection. A retrospective review was performed of all biopsied pancreatobiliary strictures at our institution over 5 years. UroVysion was unquestionably the most sensitive method and all methods were highly specific. Sensitivity was highest while maintaining specificity when a malignant interpretation was limited to cases with 5+ cells with the same polysomic signal pattern and/or loss of one or both 9p21 signals. Only UroVysion detected the metastases and a neuroendocrine tumor. In reviewing and analyzing the signal patterns, we noticed trends according to location and diagnosis. Herein we describe our method for analyzing signal patterns and propose cutoff criteria based upon observations gleaned from such analysis.

Cytogenetic monitoring of peripheral blood lymphocytes from medical radiation professionals occupationally exposed to low-dose ionizing radiation.

In order to assess the health risk of low-dose radiation to radiation professionals, monitoring is performed through chromosomal aberration analysis and micronuclei (MN) analysis. MN formation has drawbacks for monitoring in the low-dose range. Nucleoplasmic bridge (NPB) analysis, with a lower background level, has good dose-response relationships at both high and relatively low dose ranges. Dicentric and ring chromosomes were analyzed in 199 medical radiation professionals, and NPB/MN yields were analyzed in 205 radiation professionals. The effects of sex, age of donor, types of work, and length of service on these cytogenetic endpoints were also analyzed. The yields of the three cytogenetic endpoints were significantly higher in radiation professionals versus controls. Frequencies of dicentric plus ring chromosomes were affected by length of service. NPB frequencies were influenced by type of work and length of service. MN yields were affected not only by types of work and length of service but also by donor sex and age. In conclusion, dicentric plus ring chromosomes, NPB, and MN can be induced by low-dose radiation in radiation professionals. NPB is a potential biomarker to assess the health risk of occupational low-dose radiation exposure.

Establishment and multiparametric-cytogenetic validation of 60Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies.

Exposure to ionizing radiation is unavoidable to our modern developing society as its applications are widespread and increasing with societal development. The exposures may be planned as in medical applications or may be unplanned as in occupational work and radiological emergencies. Dose quantification of planned and unplanned exposures is essential to make crucial decisions for management of such exposures. This study aims to establish ex-vivo dose-response curve for 60Co-gamma-ray induced gamma-H2AX-foci by immunofluorescence using microscopy and flowcytometry with human lymphocytes. This technique has the potential to serve as a rapid tool for dose estimation and triage application during small to large scale radiological emergencies and clinical exposures. Response curves were generated for the dose range 0-4 Gy (at 1, 2, 4, 8, 16, 24, 48, 72 and 96 h of incubation after irradiation) with microscopy and 0-8 Gy (at 2, 4, 8, 16 and 24 h of incubation after irradiation) with flow cytometry. These curves can be applied for dose reconstruction when post exposure sampling is delayed up to 96 h. In order to evaluate Minimum Detection Limit (MDL) of the assay, variation of background frequency of gamma-H2AX-foci was measured in 12 volunteers. To understand the application window of the assay, gamma-H2AX foci decay kinetics has been studied up to 96 h with microscopy and response curves were generated from 1 to 96 hours post exposure. Gamma-H2AX fluorescence intensity decay kinetics was also studied up to 96 h with flow cytometry and response curves were generated from 2 to 24 hours post irradiation. Established curves were validated with dose blinded samples and also compared with standard cytogenetic assays. An inter-comparison of dose estimates was made among gamma-H2AX assay, dicentric aberrations and reciprocal translocations for application window in various dose ranges and time of blood collection after exposures.

Analytical and clinical performance of chromosomal microarrays compared with FISH panel and conventional karyotyping in patients with chronic lymphocytic leukemia.

In this single center retrospective analysis on 102 CLL patients, we assessed analytical and clinical performance of CMA against a targeted FISH panel (ATM, TP53, CEP12, D13S319 and LAMP1 loci) and karyotyping. CMA yielded additional information compared to karyotype in 39 cases (38 %). On the other hand, while CMA detected aberrations were also detected by FISH in all 31 cases (30 %), aberrations with low clonal size (<30 %) detected by FISH were missed by CMA. When evaluated with National Cancer Center Network (NCCN) guidelines, the capture rate of prognostic relevant cytogenetic information for FISH only, FISH + Chromosomes and FISH + CMA analyses were 95, 96 and 100 % respectively. With Cancer Cytogenomics Consortium (CGC) Criteria, these figures for FISH only, FISH + Chromosomes and FISH + CMA were 88 %, 92 and 100 % respectively. In conclusion, CMA provides additional analytical information to FISH and karyotyping, but this information has a clinical utility only in a small number of patients. Limit of detection (LOD) issues preclude replacement of FISH by CMA, but CMA may be a viable alternative to karyotyping. Further research is warranted.

Clinical utility of next generation sequencing to detect IGH/IL3 rearrangements [t(5;14)(q31.1;q32.1)] in B-lymphoblastic leukemia/lymphoma.

The t(5;14)(q31.1;q32.1) associated with B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is a rare, recurrent genetic abnormality recognized as a distinct entity by the 2017 World Health Organization (WHO) classification. In these cases, the IGH enhancer region (14q32.1) is juxtaposed to the vicinity of the IL3 gene (5q31.1), resulting in increased production of interleukin-3 (IL3) and subsequently a characteristic reactive eosinophilia. B-ALL with t(5;14)(q31.1;q32.1) may have a low lymphoblast count that can complicate detection of t(5;14)(q31.1;q32.1) by conventional chromosome studies. We have identified four patients with IGH/IL3 rearrangements despite normal conventional chromosome studies in each case [one patient had a non-clonal t(5;14)(q31;q32) finding]. Fluorescence in situ hybridization utilizing a laboratory-developed IGH break-apart probe set identified IGH rearrangements in three of four cases, and a next generation sequencing (NGS) based assay, mate-pair sequencing (MPseq), was required to characterize the IGH/IL3 rearrangements in each case. Three patients demonstrated a balanced t(5;14)(q31.1;q32.1) while one patient had a cryptic insertion of the IL3 gene into the IGH region. These results demonstrate that NGS-based assays, such as MPseq, confer an advantage in the detection of IGH/IL3 rearrangements that are otherwise challenging to characterize by traditional cytogenetic methodologies.