Chronic Myeloid Leukemia (CML) is defined by the reciprocal translocation t (9;22) (q34; q11.2), which produces the BCR-ABL fusion oncogene. Rare variant translocations involving additional chromosomes such as t (2;9;22) (q21; q34; q11.2) add complexity to diagnosis and management. We report a 53-year-old male with CML in chronic phase harboring this rare variant, confirmed through conventional GTG-banded karyotyping and fluorescence in situ hybridization (FISH). Clinical presentation, laboratory findings and detailed cytogenetic analysis are discussed with reference to current literature.

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative neoplasm arising from the reciprocal chromosomal translocation t (9;22) (q34; q11.2) that produces the BCR-ABL fusion gene a molecular hallmark detected in more than 90% of cases worldwide (Pablo Romero-Morelos et al., 2024). The BCR-ABL fusion gene encodes a constitutively active tyrosine kinase, which disrupts normal cellular processes by promoting uncontrolled cell proliferation, resistance to programmed cell death (apoptosis), and altered adhesion and differentiation in hematopoietic stem and progenitor cells (Stephanie Bauer et al., 2012). This kinase activity drives the expansion of the leukemic clone and underlies the pathophysiology of CML. However, the classic two-way translocation between chromosome 9 and 22 is predominant, about 5 to 10 percent of patient’s exhibit variant or complex Philadelphia chromosomes involving additional chromosomes (Ana Valencia et al., 2009). Among these, the rare three-way translocation t (2;9;22) (q21; q34; q11.2) is of special interest as it introduces complexity in chromosomal rearrangements complicating diagnosis and monitoring but retains the crucial BCR-ABL fusion as the leukemogenic driver (Madhavi G. et al., 2007).

This variant involves chromosome 2 at the q21 band in addition to the canonical breakpoints on chromosomes 9 and 22. Its presence may indicate additional genomic instability or altered regulation of genes proximal to 2q21, though the principal pathogenesis remains driven by BCR-ABL fusion kinase activity (Walid A.C. et al., 2010). Identifying these variants requires a comprehensive diagnostic approach using conventional GTG-banded karyotyping to visualize the broader chromosomal architecture combined with fluorescence in situ hybridization (FISH) for precise localization of BCR and ABL loci in complex rearrangements (Prachi S. et al., 2020).

The BCR (Breakpoint Cluster Region) gene on chromosome 22q11.2 plays a central role in Chronic Myeloid Leukemia (CML) through its fusion with the ABL1 gene, located on chromosome 9q34. This translocation, t (9;22) (q34; q11.2), creates the BCR-ABL fusion oncogene (Mohamed El-Tanani et al., 2017). The breakpoint in BCR typically occurs in the major breakpoint cluster region (M-bcr), involving exons 13 or 14, which fuse to ABL1’s exon 2. The resulting fusion protein, with constitutive tyrosine kinase activity, promotes leukemogenesis by dysregulating cell proliferation, apoptosis, and differentiation pathways (Gustavo P.A.M. et al., 2022).

The ABL1 gene encodes a non-receptor tyrosine kinase that normally regulates cell growth and survival (Pablo Romero-Morelos et al., 2024). Fusion with BCR leads to its unregulated activation, altering multiple signaling pathways such as RAS/MAPK, PI3K/AKT, and JAK/STAT, which contribute to malignant transformation (Christian Boni et al., 2022).

In the variant t (2;9;22) (q21; q34; q11.2) translocation, chromosome 2q21 is implicated as the third partner. Though not fully characterized, the involvement of 2q21 usually reflects structural chromosomal abnormalities or regulatory region disruptions rather than a specific oncogene fusion (Muhammad Asif et al., 2021). This addition may enhance genomic instability and influence disease progression, but the primary pathogenic driver remains the BCR-ABL1 fusion kinase. Overall, while chromosome 2q21 adds complexity, the fusion of BCR and ABL1 genes and their oncogenic protein product are fundamental to CML pathogenesis and progression (Federica L. et al., 2019). Furthermore, quantitative molecular assessment through real-time PCR standardized to the international scale is essential for precise monitoring of treatment response and detection of minimal residual disease. This is particularly necessary in variant translocations, where classical cytogenetic markers may be masked or obscured. Regular molecular surveillance guides timely therapeutic decisions and prognostication (Heyang Z. et al., 2021).

This case report presents a patient diagnosed with chronic phase CML bearing the rare variant t (2;9;22) (q21; q34; q11.2), detailed through integrated clinical evaluation, morphological review, cytogenetic testing, and molecular diagnostics. It highlights the critical role of comprehensive diagnostics in informing treatment strategies and monitoring outcomes in complex presentations of CML.

A 53-year-old male patient, presented to the Gujarat Cancer Research Institute (GCRI) Medical Unit-I as a known case of Chronic Myeloid Leukemia (CML) in chronic phase with previous diagnosis and treatment initiated elsewhere. The patient was clinically stable with no hepatomegaly, splenomegaly, or lymphadenopathy detected on examination.

Hematology Findings: Serial peripheral blood analyses over several months showed consistent mild anemia with hemoglobin ranging 13.3 to 15.2 g/dL, persistent leukocytosis with WBC counts up to 16.14 × 10^3/µL, and platelet counts within or modestly above reference range (332–351 × 10^3/µL). Peripheral smear findings were typical of CML, demonstrating granulocytic leukocytosis with myeloid left shift and basophilia (up to 7%). Erythrocytes displayed normocytic normochromic features.

Bone Marrow Examination:  Bone marrow biopsy conducted at GCRI revealed markedly hypercellular marrow (approximately 90?llularity) with predominant myeloid hyperplasia, substantially increased myeloid to erythroid ratio (6:1), and suppressed erythropoiesis. Megakaryocytes were increased in both number and displayed dwarf morphology. Reticulin staining showed no fibrosis, classified as MF-0. These hematopathological features were consistent with chronic phase CML.

RT– qPCR Findings: The RT-qPCR analysis showed a high BCR-ABL fusion gene load with 232,593 copies per µg RNA and a BCR-ABL/C-ABL ratio of 95.91%, corresponding to an International Scale value of 65.89%. This indicates no molecular response, as values above 0.1% reflect active disease. Limitations include assay variability and challenges in detecting rare variants, so results should be interpreted alongside clinical and cytogenetic data. Continuous molecular monitoring is essential for guiding treatment decisions in CML.

Given the complex clinical and hematologic scenario, further cytogenetic and molecular evaluation was pursued including conventional GTG banding and fluorescence in situ hybridization (FISH) targeted to BCR and ABL genes for precise characterization of chromosomal rearrangements and fusion gene confirmation. 

Cytogenetic Evaluation:

Bone marrow aspirate and peripheral blood samples were collected and cultured using standard approaches. GTG banding technique was utilized to produce characteristic chromosome banding patterns after trypsin and Giemsa staining. At least 20 high-quality metaphase spreads were comprehensively analyzed in accordance with International System for Human Cytogenomic Nomenclature (ISCN) 2024 guidelines to identify and characterize chromosomal abnormalities. 

Fluorescence In Situ Hybridization (FISH):

To detect and confirm the presence of the BCR-ABL fusion gene in the context of suspected variant Philadelphia chromosome, a dual-color FISH assay was performed on interphase nuclei and metaphase spreads using fluorescent probes directed against the BCR gene on chromosome 22q11.2 (green signals) and the ABL gene on chromosome 9q34 (orange signals). Signal patterns were interpreted to establish presence and configuration of fusion signals indicative of gene rearrangements.

Whole Chromosome Painting:

WCP FISH was done to confirm the nature of chromosomal translocations. WCP probes for chromosome 9 (green) and chromosome 2 (orange) were used on metaphase spreads (XCP Metasystems Probes, Altlußheim, Germany).

Cytogenetic Findings

Conventional cytogenetics revealed a uniform three-way translocation involving chromosomes 2, 9, and 22: 46, XY, t (2;9;22) (q21; q34; q11.2) in all analyzed metaphases. The complex rearrangement demonstrated the involvement of chromosome 2q21 in addition to the canonical 9q34 and 22q11.2 breakpoints (Figure 1). 

Figure 1: Representative images of Conventional cytogenetic results of GTG banded karyotype showing 46, XY, t (2;9;22) (q21; q34; q11.2)

FISH analysis detected fusion signals consistent with BCR-ABL rearrangement, presenting a variant signal pattern of 2 green signals and 1 orange signal to 1 fusion signal in 100% of cells, confirming the functional fusion gene within the context of the complex rearrangement (Figure 2b). 

Figure 2b: BCR-ABL rearrangement, presenting a variant signal pattern of 2 green signals and 1 orange signal to 1 fusion signal in 100% of cells using FISH Technique.

Whole Chromosome Painting (WCP) analysis of the metaphase spread revealed that chromosomal material from chromosome 2 (visualized as orange fluorescence) was present on chromosome 9 (visualized as green fluorescence). This colocalization of fluorescent signals provides definitive evidence of a translocation between chromosomes 2 and 9. The observed fusion of orange and green signals on a single chromosome segment scientifically confirms the presence of complex chromosomal rearrangement, supporting the diagnosis of a t(2;9) translocation at the molecular cytogenetic level (Figure 2a).

Figure 2a: Whole Chromosome Painting (WCP) analysis showed orange fluorescence from chromosome 2 on chromosome 9 (green), indicating chromosomal material exchange.

The fused orange-green signals confirmed a complex t (2;9) translocation at the molecular cytogenetic level.

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell malignancy chiefly characterized by the reciprocal chromosomal translocation t (9;22) (q34; q11.2), termed the Philadelphia (Ph) chromosome (Mohamed El-Tanani et al., 2017). This translocation leads to the formation of the BCR-ABL fusion oncogene, which encodes a constitutively active tyrosine kinase that drives unchecked proliferation and resistance to apoptosis in myeloid progenitor cells, underpinning the pathogenesis of CML (Ruibano R. 2005). While the classical two-way Ph translocation is observed in over 90% of CML cases globally, approximately 5–10% of patients manifest variant or complex Philadelphia chromosome translocations involving additional chromosomes (Ana I. et al., 2021). These complex rearrangements contribute significant challenges in diagnosis, molecular monitoring, and potentially prognosis (Madhavi G. et al., 2007).

One such rare and clinically significant variant is the three-way translocation t (2;9;22) (q21; q34; q11.2), where chromosome 2 at band q21 participates alongside chromosomes 9 and 22 (Diwakar S. et al., 2024). The pathogenic relevance of the 2q21 breakpoint remains unclear but is thought to involve genomic instability or altered chromatin conformation, rather than a direct oncogenic event. Identifying such variant translocations necessitates integrated cytogenetic and molecular diagnostics to ensure precise characterization and management (Chauffaille, M. deL., et al., 2015). Conventional Giemsa-trypsin-Giemsa (GTG) banding remains a foundational method to visualize chromosomal architecture and detect large-scale rearrangements. However, its limitations become prominent with complex or cryptic translocations where breakpoints may be subtle or masked (Xiaoxi Z. et al., 2025). Complementary fluorescence in situ hybridization (FISH) targeting the BCR and ABL loci enables direct detection of fusion gene signals, clarifying variant rearrangements unseen by karyotyping alone (R S Verma et al., 1982).

The pathogenesis of Chronic Myeloid Leukemia (CML) is centered around the formation of the Philadelphia chromosome, arising from a reciprocal translocation between the long arms of chromosomes 9 and 22, t (9;22) (q34; q11.2) (Stephanie Bauer et al., 2012). This rearrangement juxtaposes the Breakpoint Cluster Region (BCR) gene on chromosome 22q11.2 and the Abelson proto-oncogene (ABL1) gene on chromosome 9q34, generating the BCR-ABL fusion gene, the principal molecular driver of CML (Hossein A et al., 2018). 

BCR gene (22q11.2): The BCR gene serves as the critical partner in the translocation. It contains multiple breakpoint cluster regions, the most common being the major breakpoint cluster region (M-bcr), located between exons 12 and 16. The typical fusion transcripts, such as e13a2 or e14a2, result in the p210 BCR-ABL fusion protein prevalent in most CML patients (C M Croce et al., 1987). These fusion proteins exhibit constitutive tyrosine kinase activity, driving oncogenic signaling pathways. Alternative breakpoints in minor or micro BCR regions give rise to variant protein isoforms associated with different leukemic phenotypes (Takashi I. et al., 1993). ABL1 gene (9q34): The ABL1 gene encodes a non-receptor tyrosine kinase involved in normal cellular functions including proliferation, differentiation, and apoptosis. Translocation with BCR fundamentally alters ABL1 regulation, producing an oncogenic fusion kinase with persistent activity (Jean Y.J, 2014). This active kinase deregulates signaling cascades such as RAS/MAPK, PI3K/AKT, and JAK/STAT, leading to enhanced cell survival and growth, genomic instability, and leukemic transformation. Breakpoints within ABL1 generally occur between exon 1a and exon 2, but rare variations skipping or involving other exons have been documented, influencing protein structure and function (Pooja C. et al., 2024).

Chromosome 2q21 region: The variant t (2;9;22) (q21; q34; q11.2) incorporates a third chromosomal breakpoint at 2q21. Unlike BCR and ABL1, the genes or specific sequences involved in 2q21 are less clearly defined or consistent (Chauffaille, M. deL., et al., 2015). Most studies suggest that 2q21 involvement is structural and may represent non-coding or regulatory elements rather than an expressed fusion partner. However, this chromosomal involvement increases the complexity of the rearrangement and may contribute to genomic instability and epigenetic modifications, thereby potentially influencing clonal evolution and resistance mechanisms (Meghana R. et al., 2024). Current evidence supports that the dominant oncogenic driver remains the BCR-ABL fusion gene, with 2q21 acting mainly as a bystander or modifier of chromosomal architecture (Pooja C. et al., 2024). 

This structural complexity highlights the need for advanced cytogenetic techniques such as fluorescence in situ hybridization (FISH) and whole chromosome painting (WCP) to fully characterize such aberrations (Ping He et al., 2022). Furthermore, integration with molecular diagnostics ensures comprehensive disease profiling that guides therapeutic monitoring and prognosis. Molecular assessment via quantitative real-time PCR (RT-qPCR) standardized to the international scale is indispensable to quantify BCR-ABL transcript levels for sensitive monitoring of therapeutic response and minimal residual disease. This molecular monitoring is especially vital in variant cases, where conventional cytogenetic results may be equivocal or incomplete (Paula Jp de Vree et al., 2009).

Clinically, patients bearing variant Ph translocations generally demonstrate treatment responses comparable to those with classical translocations when managed with tyrosine kinase inhibitors (TKIs). Nonetheless, persistent high transcript levels or acquisition of additional cytogenetic abnormalities require vigilant monitoring and could compel therapeutic escalation (Muhammad Asif et al., 2021). Further elucidation of the biological consequences of variant breakpoints, their influence on clonal evolution, and TKI resistance mechanisms remains an active area of research. Understanding these variants not only enriches cytogenetic and molecular diagnostic capabilities but also supports tailored and improved patient management strategies (Elias J. et al., 2025).

This detailed overview synthesizes current knowledge on variant Philadelphia translocations in CML, with special focus on the t (2;9;22) (q21; q34; q11.2) alteration, underscoring its diagnostic complexities and clinical implications in the modern therapeutic landscapes.

Our report emphasizes the significance of comprehensive cytogenetic investigation combined with FISH and molecular analyses for the accurate diagnosis and precise characterization of rare variant Philadelphia translocations, such as t (2;9;22) (q21; q34; q11.2), in CML. Although additional chromosomal involvement complicates the genomic landscape, the central pathogenic driver remains the BCR-ABL fusion gene. Continued molecular monitoring is indispensable for optimizing targeted therapy and ensuring improved patient outcomes in these complex presentations.