Ioannis Panagopoulos

For decades, the "death of cancer cytogenetics" has been repeatedly proclaimed and has grown louder in the era of omics. However, cytogenetics continues to evolve, retaining its central role in the study of chromosomes. From the microscopic visualization of numerical and structural aberrations using staining and banding techniques, the field has steadily incorporated new methodologies, including fluorescence in situ hybridization, array comparative genomic hybridization, high-throughput sequencing, and, most recently, optical genome mapping. These approaches have revealed unprecedented details of chromosome structure and behavior, uncovered catastrophic genomic events in cancer, and given rise to new concepts such as cytogenomics and chromosomics. Although cytogenetics is sometimes viewed as synonymous with chromosome banding, its scope, namely the study of chromosome structure and behavior, extends far beyond any single method. Chromosomes remain indispensable for understanding genome architecture, chromosomal instability, and fusion-gene mechanisms in neoplasia, irrespective of the technologies applied. Rooted in its past yet continually branching outwards with each scientific advance, today's cytogenetics integrates classical and modern approaches, carrying forward the legacy of chromosome banding while adopting genome-wide technologies. These developments underscore the enduring importance of cytogenetics for interpreting the structural complexity of cancer genomes. This review aims to demonstrate that cytogenetics, far from being obsolete, remains a vital component of modern cancer research by highlighting essential insights into chromosome structure, genome architecture, and fusion-gene mechanisms in the era of -omics.

Background: Leiomyomas of the gastrointestinal tract (GI) are benign smooth muscle neoplasms with limited genetic characterization. Molecular investigations may improve diagnostic classification and enhance understanding of their biological behavior. Methods: RNA sequencing using multiple fusion-detection algorithms was performed on an ileal leiomyoma. Key findings were validated by RT-PCR and Sanger sequencing. Results: A MYH11::GLI3 fusion was identified. Additional chimeric transcripts were detected but interpreted as secondary events based on limited read support. The biological relevance of MYH11::GLI3 relates to smooth muscle specific MYH11 expression and GLI3 -mediated Hedgehog signaling. Conclusion: This study reports, for the first time, the identification of a MYH11::GLI3 chimera in gastrointestinal leiomyoma, thereby expanding the molecular spectrum of these tumors. Deregulation of GLI3 may represent an alternative mechanism of Hedgehog pathway perturbation in this neoplasm. The frequency and clinical significance of GLI3-rearranged gastrointestinal smooth muscle tumors remain to be determined.

Background: Benign fibro-osseous lesions are characterized by the replacement of normal bone with cellular fibrous connective tissue with new bone formation. The published cytogenetic information on these tumors is limited to only few cases. Here, we report the cytogenetic and molecular genetic findings of a fibro-osseous tumor.Methods: A fibro-osseous lesion was investigated for genetic abnormalities using banding cytogenetics, fluorescence in situ hybridization (FISH), RNA sequencing, and direct cycle Sanger sequencing.Results: The karyotype was 46,XX,t(4;11;14;12)(q35;p15;q22;q13)[7]/46,XX [3], with no rearrangement of HMGA2. RNA sequencing revealed two FRMD6::PTH chimeric transcripts, originating from the fusion point 14q22;11p15 of the t(4;11;14;12). In these transcripts, exon 1 of FRMD6 fused to either exon 1 or exon 2 of PTH. Direct cycle sequencing confirmed the presence of these FRMD6::PTH chimeric transcripts.Conclusion: This study reports, for the first time, the presence of the FRMD6::PTH chimera in fibro-osseous tumor. In this chimera the expression of the entire coding region of PTH is regulated by the ubiquitously expressed FRMD6 gene promoter. Dysregulation of PTH expression may have significant implications for processes regulated by PTH protein.

Background/Aim: Myelodysplastic syndromes (MDSs) are clonal bone marrow disorders characterized by ineffective hematopoiesis. They are classified based on morphology and genetic alterations, with SF3B1 variants linked to favorable prognosis and MECOM rearrangements associated with poor outcomes. The combined effects of these alterations remain unclear. We report an MDS patient carrying both SF3B1 and MECOM alterations who developed transient eosinophilia accompanied by a TNIP1::PDGFRB chimera in a subset of MECOM-affected cells. Case Report: A 73-year-old woman was diagnosed with myeloid neoplasia with excess blasts and multilineage dysplasia (MDS-EB1). Six months later, SF3B1 mutations were identified, leading to a diagnosis of MDS-SF3B1. Despite azacitidine treatment, her condition worsened, showing hypercellular bone marrow and eosinophilia. Genetic analysis revealed a t(2;3)(p15~23;q26)/MECOM rearrangement and TNIP1::PDGFRB chimera. Imatinib eradicated eosinophilia and reduced TNIP1::PDGFRB-positive cells, but the MECOM-clone persisted. Subsequent treatments, including hydroxyurea, mercaptopurine, and low-dose cytarabine, were ineffective. FLT3 mutations and high EVI1 transcript levels were later detected. The patient succumbed to progressive disease. Conclusion: This case highlights the complexity of MDS and the importance of genetic abnormalities in treatment planning. Persistent MECOM rearrangement and the TNIP1::PDGFRB chimera emphasize the need for further research into resistance mechanisms.

Background/Aim: The term “calcified chondroid mesenchymal neoplasm” was introduced in 2021 to describe a group of tumors characterized by various morphological features, including the formation of cartilage or chondroid matrix. These tumors frequently carry chimeric genes where the 5′-end partner gene is fibronectin 1 and the 3′-end partner gene codes for receptor tyrosine kinase. Our study explores fusion of the genes platelet-derived growth factor receptor alpha (PDGFRA) and ubiquitin-specific peptidase 8 (USP8) in calcified chondroid mesenchymal neoplasm. Case Report: Genetic investigations were conducted on a tumor located in the leg of a 71-year-old woman. G-banding analysis of short-term cultured tumor cells revealed the karyotype 46,XX,t(4;15)(q12;q21)[6]/46,XX[4]. RNA sequencing detected in-frame PDGFRA::USP8 and USP8::PDGFRA chimeric transcripts, which were validated by RT-PCR/Sanger sequencing. The PDGFRA::USP8 chimeric protein is predicted to have cell membrane location and functions as a chimeric ubiquitinyl hydrolase. The USP8::PDGFRA protein was predicted to be nuclear and function as a positive regulator of cellular metabolic process. Conclusion: We report, for the first time, a calcified chondroid mesenchymal neoplasm carrying a balanced t(4;15)(q12;q21) chromosomal translocation, resulting in the generation of both PDGFRA::USP8 and USP8::PDGFRA chimeras. The PDGFRA::USP8 protein is located on the cell membrane and functions as a chimeric ubiquitinyl hydrolase, activated by PDGFs. Conversely, USP8::PDGFRA is a nuclear protein regulating metabolic processes.

Background/Aim: Constitutional chromosomal aberrations are rare in hematologic malignancies and their pathogenetic role is mostly poorly understood. We present a comprehensive molecular characterization of a novel constitutional chromosomal translocation found in two siblings – sisters – diagnosed with myelodysplastic syndrome (MDS). Materials and Methods: Bone marrow and blood cells from the two patients were examined using G-banding, RNA sequencing, PCR, and Sanger sequencing. Results: We identified a balanced t(17;19)(q21;p13) translocation in both siblings’ bone marrow, blood cells, and phytohemagglutinin-stimulated lymphocytes. The translocation generated a MYO1F::WNK4 chimera on the der(19)t(17;19), encoding a chimeric serine/threonine kinase, and a VPS25::MYO1F on the der(17), potentially resulting in an aberrant VPS25 protein. Conclusion: The t(17;19)(q21;p13) translocation found in the two sisters probably predisposed them to myelodysplasia. How the MYO1F::WNK4 and/or VPS25::MYO1F chimeras, perhaps especially MYO1F::WNK4 that encodes a chimeric serine/threonine kinase, played a role in MDS pathogenesis, remains incompletely understood.

Background/Aim: In precursor B-cell lineage acute lymphoblastic leukemia (BCP-ALL), leukemic cells harbor genetic abnormalities that play an important role in the diagnosis, prognosis, and treatment. A subgroup of BCP-ALL is characterized by the presence of a Philadelphia (Ph) chromosome and a chimeric BCR::ABL1 gene, whereas in another subgroup, leukemic cells exhibit near-haploidy with chromosome number 24-30. This study presents the third documented case of BCP-ALL in which a near haploid clone concurrently displayed a Ph chromosome/BCR::ABL1. Case Report: Bone marrow cells obtained at diagnosis from a 25-year-old man with BCP-ALL were genetically investigated using G-banding, fluorescence in situ hybridization, and array comparative genomic hybridization. Leukemic cells had an abnormal karyotype 28,X,−Y,+6,+10,+18,+21,+ der(22) t(9;22)(q34;q11)[13]/28,idem, del(10)(q24),der(12) t(1;12) (q21;p13)[2]/46,XY[3], retained heterozygosity of the disomic chromosomes 6, 10, 18, and 21, had breakpoints in introns 1 of ABL1 and BCR, and carried a BCR::ABL1 chimera encoding the 190 kDa BCR::ABL1 protein. Conclusion: The coexistence of the BCR::ABL1 chimera and near-haploidy in the same cytogenetic clone suggested a possible synergistic role in leukemogenesis, with the former activating signaling pathways and the latter disrupting gene dosage balance.

Introduction: Intramuscular myxomas are benign tumors that are challenging to diagnose, especially on core needle biopsies. Acquired chromosomal aberrations and pathogenic variants in codon 201 or codon 227 in GNAS complex locus gene (GNAS) have been reported in these tumors. Here we present our genetic findings in a series of 22 intramuscular myxomas.Materials and methods: The tumors were investigated for the presence of acquired chromosomal aberrations using G-banding and karyotyping. Pathogenic variants in codon 201 or codon 227 of GNAS were assessed using direct cycle Sanger sequencing and Ion AmpliSeq Cancer Hotspot Panel v2 methodologies.Results: Eleven tumors carried chromosomal abnormalities. Six tumors had numerical, four had structural, and one had both numerical and structural chromosomal aberrations. Gains of chromosomes 7 and 8 were the most common abnormalities being found in five and four tumors respectively. Pathogenic variants in GNAS were detected in 19 myxomas (86%) with both methodologies. The detected pathogenic variants were p.R201H in nine cases (seven with abnormal and two with normal karyotypes), p.R201C in five cases, all with normal karyotypes, p.R201S in three cases (two with abnormal and one with normal karyotype), p.R201G in one case with a normal karyotype, and p.Q227E in one case with a normal karyotype.Conclusion: Firstly, our data indicate a possible association between chromosomal abnormalities and GNAS pathogenic variants in intramuscular myxomas. Secondly, the presence of the rare pathogenic variants R201S, p.R201G and p.Q227E in 26% (5 out of 19) of myxomas with GNAS pathogenic variants shows that methodologies designed to detect only the common “hotspot” of p.R201C and p.R201H will give false negative results. Finally, a comparison between Ion AmpliSeq Cancer Hotspot Panel v2 and direct cycle Sanger sequencing showed that direct cycle Sanger sequencing provides a quick, reliable, and relatively cheap method to detect GNAS pathogenic variants, matching even the most cutting-edge sequencing methods.

Background/Aim: Mixed phenotype acute leukemia (MPAL) is a rare hematologic malignancy in which the leukemic cells cannot be assigned to any specific lineage. The lack of well-defined, pathogenetically relevant diagnostic criteria makes the clinical handling of MPAL patients challenging. We herein report the genetic findings in bone marrow cells from two pediatric MPAL patients. Patients and Methods: Bone marrow cells were examined using G-banding, array comparative genomic hybridization, RNA sequencing, reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization. Results: In the first patient, the genetic analyses revealed structural aberrations of chromosomal bands 8p11, 10p11, 11q21, and 17p11, the chimeras MLLT10::PICALM and PICALM::MLLT10, and imbalances (gains/losses) on chromosomes 2, 4, 8, 13, and 21. A submicroscopic deletion in 21q was also found including the RUNX1 locus. In the second patient, there were structural aberrations of chromosome bands 1p32, 8p11, 12p13, 20p13, and 20q11, the chimeras ETV6::LEXM and NCOA6::ETV6, and imbalances on chromosomes 2, 8, 11, 12, 16, 19, X, and Y. Conclusion: The leukemic cells from both MPAL patients carried chromosome aberrations resulting in fusion genes as well as genomic imbalances resulting in gain and losses of many gene loci. The detected fusion genes probably represent the main leukemogenic events, although the gains and losses are also likely to play a role in leukemogenesis.