Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder caused by the BCR::ABL1 fusion gene, resulting from a reciprocal translocation between chromosomes 22 and 9. Quantification of BCR::ABL1 transcript levels in peripheral blood by RT-qPCR represents the gold standard for molecular response (MR) monitoring, providing essential clinical information on treatment efficacy. Xpert^® BCR-ABL Ultra is a fully automated in vitro diagnostic test that quantitatively detects e13a2 and e14a2 BCR::ABL1 transcripts using a single-use cartridge that integrates RNA extraction, cDNA synthesis, nested real-time PCR, and signal detection within a rapid, closed, and user-friendly system. In this study, we evaluated Xpert^® BCR-ABL Ultra as an alternative to validated systems currently used by four highly specialized Italian laboratories affiliated with the Italian national laboratory network for CML. A total of 129 peripheral blood samples from CML patients at various disease stages, along with two external quality control materials, were analyzed. We assessed the test’s repeatability, specificity, and stability. Concordance of BCR::ABL1%IS values generated by the different methods was evaluated using EUTOS criteria and Bland–Altman analysis. Finally, MR value concordance was analyzed based on European LeukemiaNet recommendations or calculated using the formula 2 − log₁₀(BCR::ABL1%IS). Xpert^® BCR-ABL Ultra demonstrated high repeatability and stability. The BCR::ABL1%IS values obtained with this assay showed strong concordance with those generated by local reference methods, and MR classifications were consistent across platforms. These findings confirm the robustness, accuracy, and efficiency of the Xpert^® BCR-ABL Ultra assay, supporting its use as a reliable alternative to currently validated systems for the routine clinical monitoring of CML patients.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases originating from hematopoietic stem cell transformation, characterized by the clonal proliferation of hematopoietic progenitors. A specific subset includes myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase (TK) gene fusions, particularly involving PDGFR A or B, which are sensitive to TK inhibitor treatment. We report a case of a 21-year-old patient with a myeloproliferative/myelodysplastic neoplasm, presenting with hyperleukocytosis, anemia, thrombocytopenia, and elevated LDH. The peripheral blood smear showed hypogranular neutrophils, eosinophils, basophils, and myeloid precursors. The absence of BCR::ABL1 and mutations in JAK2, CALR, and MPL excluded common MPNs. Cytogenetic analysis revealed a rearrangement between chromosomes 5 and 14. FISH analysis confirmed an inverted insertion from chromosome 5 to chromosome 14, involving the PDGFRB gene. WGS and RNAseq identified a fusion between PDGFRB and CCDC88C, causing the constitutive activation of PDGFRB. The fusion gene was confirmed by sequencing. This allowed for targeted therapy with a tyrosine kinase inhibitor (TKI), leading to molecular remission monitored by RT-qPCR. This case highlights how a multidisciplinary approach can identify atypical transcripts in MPN, guiding targeted therapy with TK inhibitors, thus resulting in effective treatment and molecular remission.

Acute myeloid leukaemia (AML) is a genetically heterogeneous haematologic malignancy driven by the stepwise acquisition of chromosomal abnormalities and somatic mutations, resulting in clonal evolution. Current guidelines recommend integrating morphology, immunophenotyping, cytogenetics, and molecular analyses for disease classification and prognostic stratification. However, discrepancies between conventional cytogenetics and genome-wide approaches may occur. We describe a 70-year-old man presenting with severe pancytopenia, diagnosed with AML based on bone marrow morphology and immunophenotyping. Karyotyping revealed a deletion of chromosome 11 and additional material on chromosome 12. Targeted Next-Generation Sequencing identified pathogenic variants in FLT3, SF3B1, and EZH2, together with copy number losses involving chromosomes 7 and 11, the former not detected by chromosome banding analysis. To resolve this discrepancy, genome-wide Array-CGH+SNP analysis confirmed monosomy 7 and uncovered additional genomic abnormalities. Fluorescence in situ hybridization (FISH) validated chromosome 7 loss at the single-cell level. Taken together, these findings suggest that monosomy 7 was restricted to a leukaemic subclone underrepresented in metaphase analysis, likely owing to differential proliferative capacity among leukaemic populations. This case highlights how integrated cytogenomic profiling can uncover clinically relevant clonal heterogeneity missed by conventional cytogenetics alone, refining ELN risk stratification and underscoring the value of combining chromosome-based and DNA-based technologies in AML.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases originating from hematopoietic stem cell transformation, characterized by clonal proliferation of hematopoietic progenitors. A specific subset includes myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase (TK) gene fusions, particularly involving PDGFR A or B, which are sensitive to TK inhibitor treatment. We report a case of a 21-year-old patient with a myeloproliferative/ myelodysplastic neoplasm, presenting with hyperleukocytosis, anemia, thrombocytopenia, and elevated LDH. Peripheral blood smear showed hypogranular neutrophils, eosinophils, basophils, and myeloid precur-sors. Absence of BCR::ABL1 and mutations in JAK2, CALR, and MPL ex-cluded common MPNs. Cytogenetic analysis revealed a rearrangement between chromosomes 5 and 14. FISH analysis confirmed an inverted insertion from chromosome 5 to chromosome 14, involving the PDGFRB gene. WGS and RNAseq identified a fusion between PDGFRB and CCDC88C, causing constitutive activation of PDGFRB. The fusion gene was confirmed by sequencing. This allowed for targeted therapy with a tyrosine kinase inhibitor (TKI), leading to molecular remission monitored by RT-qPCR. This case demonstrates how a multidisciplinary approach can identify atypical transcripts in MPN, guiding targeted therapy with TK inhibitors, resulting in effective treatment and molecular remission.

Next-generation sequencing (NGS) clonality assessment has the potential to overcome the conventional limitations of PCR-based methods. This study aimed at comparing the performance and concordance of the EuroClonality-NGS approach and the LymphoTrack^® Dx assay for immunoglobulin (IG)/T-cell receptor (TR)-marker identification at diagnosis. Across four quality-control rounds, six Italian laboratories analyzed 23 acute lymphoblastic leukemia (ALL) and five chronic lymphocytic leukemia (CLL) cases. Overall, 171 rearrangements were identified; 80.7% were detected by both methods. Among shared targets, the concordance was 89.1%, with a higher agreement for IG (IGH 100%–IGK 92.1%) than for TR loci (TRG 78.9%–TRB 81.8%). Bland–Altman analysis indicated no statistically significant systematic bias between methods [mean bias 1.99% (95% CI: −5.29 to +1.31%)]. The Spearman correlation was ρ = 0.680 (p < 0.001). Discordances (10.9%) rarely yielded suitable sensitive PCR assays for disease monitoring (20%) and were never the sole marker in individual patients. In larger independent cohorts, similar rates of no-marker cases were observed (~4–5% in adults), with higher frequencies in T-ALL and no association with blast percentage (Spearman’s ρ = 0.447, p = 0.450 in adults and ρ = 0.700, p = 0.188 in childhood). These findings support the reliability of both methods for diagnostic screening, while highlighting locus-specific variability and the importance of multi-target identification.