Mihaela Dragomir

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and genomic profiling has become increasingly vital for risk stratification. We conducted a retrospective single-center study including 96 newly diagnosed pediatric patients with ALL and mixed phenotype acute leukemia to characterize the genomic landscape using conventional and molecular cytogenetics, multiplex ligation-dependent probe amplification and targeted next-generation sequencing and to evaluate associations with treatment response and survival. Mutation co-occurrence analysis revealed a NRAS/KRAS/JAK cluster and an ETV6::RUNX1 cluster characterized by relative mutational exclusivity, with additional associations between NOTCH1–CDK4 and CDK4–CCND3 alterations. DelCDKN2A/CDKN2B alterations showed a trend toward higher day-15 minimal residual disease (MRD) levels and inferior 2-year event free survival rate (EFS). DelIKZF1-positive cases showed lower 2-year EFS. In contrast, ETV6::RUNX1 and high hyperdiploidy were associated with favorable early response and EFS. DelPAX5 did not independently influence outcome. ZNF384 positive cases showed higher early MRD levels despite excellent survival outcomes. NRAS/KRAS mutations were significantly associated with higher positive day-15 MRD (Wilcoxon, p = 0.0067) and remained independently associated after adjustment for white blood cell count and cytogenetic subgroup. Intermediate risk (IR) and high-risk groups showed comparable 2-year EFS, indicating limited discrimination by conventional risk stratification. The IR group displayed a heterogeneous genomic profile, with NRAS/KRAS, Ph-like mutations and delCDKN2A/CDKN2B among the most frequent alterations. These observations highlight the potential of integrated genomic profiling to refine risk stratification, particularly by identifying clinically relevant subgroups within the IR category.

Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by mutations in the F8 gene, resulting in deficient or dysfunctional factor VIII (FVIII). This study aimed to characterize the mutational profile of HA in Romanian patients using next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). A total of 107 patients were analyzed, revealing pathogenic or likely pathogenic variants in 96.3% of cases. The identified mutations included missense (30.5%), nonsense (9.1%), small deletions (6.4%), small insertions (2.1%), splice-site variants (4.3%), large deletions (1.6%), and large duplications (1.1%). Large intron inversion was previously found in 37.5% of the patients. Novel variants accounted for 21.5% of identified mutations, expanding the spectrum of F8 variants in this population. This study underscores the genetic heterogeneity of HA and provides insights into genotype–phenotype correlations, aiding in clinical management and prenatal diagnosis.

Multiple myeloma is a hematologic neoplasm caused by abnormal proliferation of plasma cells. Sequencing studies suggest that plasma cell disorders are caused by both cytogenetic abnormalities and oncogene mutations. Therefore, it is necessary to detect molecular abnormalities to improve the diagnosis and management of MM. The main purpose of this study is to determine whether NGS, in addition to cytogenetics, can influence risk stratification and management. Additionally, we aim to establish whether mutational analysis of the CD138 cell population is a suitable option for the characterization of MM compared to the bulk population. Following the separation of the plasma cells harvested from 35 patients newly diagnosed with MM, we performed a FISH analysis to detect the most common chromosomal abnormalities. Consecutively, we used NGS to evaluate NRAS, KRAS, BRAF, and TP53 mutations in plasma cell populations and in bone marrow samples. NGS data showed that sequencing CD138 cells provides a more sensitive approach. We identified several variants in BRAF, KRAS, and TP53 that were not previously associated with MM. Considering that the presence of somatic mutations could influence risk stratification and therapeutic approaches of patients with MM, sensitive detection of these mutations at diagnosis is essential for optimal management of MM.

Objective: TET2 gene (4q24) is one of the most important regulatory factors of the methylation process in human genome. The homologous protein is directly involved in convertion of 5-methylcytosine into 5-hydroximethylcytosine (1). Loss of function mutations in TET2 gene are considered to be driver mutations (initiating mutations) for leukemogenesis. In some cases they can be found in early clonal cells of Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) or myelodisplastic syndromes (MDS) (2). The aim of this study is to determinate the frequency of TET2 mutations in Acute Leukemia (AL) patients. Materials and methods: This retrospective study enrolled 24 patients newly diagnosed with AML (19 patients) and ALL (5 pacients). By using Real-Time PCR we obtained a relative cuantification of TET2 gene level of expression taking ABL gene as reference. Outcomes: Low levels of TET2 gene expression were found in 6 patients (25%), 3 of them being diagnosed with AML and 3 with ALL. No cytogenetic anomalies were noticed. However, all these patients had blastocytosis and higher white blood cells count than pacients with normal levels of expression. There was one case with mutations in both TET2 gene and NPM1 gene and one case that associated a defective TET2 gene and an internal tandem duplication of FLT3-ITD. Conclusions: In our study, TET2 gene loss of function mutations occured on a specific background which involved some characteristic mutations for AL and some particular features of cell morphology. We aim to continue this study by investigating the long term effects of this epigenetic mutation in AL patients.

Background and Objectives: Despite the vast heterogeneity in the genetic defects causing hemophilia A (HA), large intron inversions represent a major cause of disease, accounting for almost half of the cases of severe HA worldwide. We investigated the intron 22 and intron 1 inversion status in a cohort of Romanian unrelated patients with severe HA. Moreover, we evaluated the role of these inversions as relative risk factors in inhibitor occurrence. Materials and Methods: Inverse shifting—a polymerase chain reaction method was used to detect the presence of intron 22 and intron 1 inversions in 156 Romanian patients with HA. Results: Intron inversion 22 was found in 41.7% of the patients, while intron 1 inversion was detected in 3.2% of the patients. Overall, large intron inversions represented the molecular defect in 44.9% of the studied patients. Our findings are in accord with previously published reports from Eastern Europe countries and with other international studies. The risk of inhibitor development was higher in patients with inversion 1 compared to the patients with HA without any inversion detected. Conclusions: The current study demonstrates the major causative role of large intron inversions in severe HA in Romanian patients. Moreover, our study confirms the contribution of intron 1 inversion in inhibitor development.

Introduction: miRNAs are involved in the pathogenesis of neoplastic syndromes by silencing target genes. As previously shown, hsa-mir-4328 is downregulated in Acute Promyelocytic Leukemia (APL). Objectives: The study aims to identify the somatic mutations of the MIR-4328 gene that caused its downregulation in APL and their localization in key regions of the mature miRNA structure. Material and methods: The study included 24 subiects: the study group (10 patients at the onset of APL, as well as at remission and relapse) and the control group (10 apparently healthy patients). High-Resolution Melting (HRM) was used for genotyping. Results: As MIR-4328 gene has not been studied before, a structure design of the coding region was performed to better understand the impact of somatic mutations on the mature miRNA. Following HRM, 2 mutant genotypes were identified, different from the wild-type (WT) genes. Conclusions and discussion: Due to the major deviation of the mutant genotypic curves compared to WT, the existence of major mutations (probably insertions/deletions) can be assumed. If these mutations are located in the seed region of the gene, attachment to exon 3 of the RARA gene is no longer possible, and therefore overexpression of the target gene occurs. Also, frameshift mutations, or substitutions that change the nucleotide sequence of the seed region, can produce a completely different mature miRNA that may have tropism for another gene. To test these hypotheses, sequencing of the entire gene is required.

The hallmark of acute promyelocytic leukemia (APL) is the presence of the characteristic fusion transcript of the promyelocytic leukemia gene with the retinoic acid receptor α gene (PML::RARA), which is a molecular target for all-transretinoic acid (ATRA) and arsenic trioxide (ATO). Therapies with ATRA plus ATO have excellent outcomes in terms of complete remission rate, overall survival, and achievement of a deep and durable molecular response with a very low incidence of relapse. However, although the combination of ATRA and ATO has lower hematologic toxicity than standard chemotherapy, its use is associated with a spectrum of unique toxicities, such as hepatic toxicity, QTc (QT interval corrected) prolongation, neurotoxicity, and differentiation syndrome. Hematologists treating patients with acute promyelocytic leukemia should be aware of possible early-complications of the ATRA plus ATO regimen and aim to balance treatment-related adverse events and efficacy. Careful follow-up of the patient’s clinical course is essential to identify and treat each complication. This study focused on treatment-related complications during induction and consolidations in patients with APL who received ATRA plus ATO. We retrospectively analyzed 26 adult patients diagnosed with APL between 2019 and 2022 at our center to identify the incidence rate, severity and time of onset of short-term non-hematologic adverse events relative to the initiation of the ATRA and ATO regimen. Additionally, we review the existing literature regarding the toxic side effects of ATO in APL and discuss how its most frequent toxicities should be managed. The median follow-up of our patients was 21.5 months (range, 1-48 months) and the most common adverse events were hepatic toxicity (69.2%), infections (46.1%), differentiation syndrome(23%) and QT prolongation (11.5%). All patients experienced at least one adverse event and grade 3 and 4 toxicities occurred in 57.6 % patients; these generally consisted of liver enzyme elevations, QTc prolongation, but also some rare manifestations such as musculoskeletal pain syndrome (1 patient) and ATO-related pancreatitis (1 patient). Most patients developed side-effects during induction. This is the first study about Romanian patients diagnosed with APL. Our results suggest that all treatment-related adverse events, except for differentiation syndrome are manageable, self-limiting and reversible. All patients, except for two (one died during induction and one died during consolidation), were able to complete the treatment consolidation with ATO and ATRA.

B-cell precursor acute lyphoblastic leukemia (ALL) is a common pediatric malignancy and patients may have significant benefits from monoclonal antibodies therapy with increased survival rates. Positive CD20 expression is identified in about half of these patients and its presence may serve as a prognostic factor in disease evolution. We performed a retrospective study including 114 patients diagnosed with B-ALL and evaluated the expression of CD20 through flow cytometry at diagnosis and on day 15. Additional immunophenotypic analyses as well as cytogenetic and molecular genetic analyses were also performed. We observed an increase in the mean fluorescence intensity (MFI) of CD20 between diagnosis—1.9 (1.2–3.26) and day 15: 6.17 (2.14–27.4), (p < 0.0001). Furthermore, we assessed that both diagnosis and day 15 CD20 MFI had an impact on RFS and OS, respectively, for cut-off values of >8.08 at diagnosis and >28.65 at day 15. In conclusion, CD20 expression appears to be a poor prognostic feature of B-ALL in pediatric patients. In this study, stratification of the outcome by the intensity of CD20 has implications concerning the allocation to rituximab-based chemotherapy and may offer new, potentially useful information for pediatric patients with B-ALL.

Introduction: Essential thrombocythemia is a chronic myeloproliferative neoplasm associated with thrombo-hemorrhagic events and the progression to myelofibrosis or acute myeloid leukemia. The purpose of this article is to present real-world data on ET cases diagnosed and managed between 1998 and 2020 in the largest, tertiary hematology reference center in Romania and to evaluate the impact of thrombotic events on survival. Methods: A real-world, retrospective cohort-type study was conducted. We collected and statistically analyzed data from 168 patients who met the 2016 WHO diagnostic criteria for ET and who were managed between 1998 and 2020 in our center. Results: The median age at diagnosis of ET was 51.8 years, with a female predominance (66.07%). The JAK2V617F mutation was detected in 60.71% of patients. Leukocytosis at diagnosis was associated with a higher risk of thrombosis, and JAK2V617F-positive cases exhibited a 1.5-fold higher risk of developing thrombotic events. The average survival in ET with major thrombosis was 14.5 years versus 20.6 years in ET cases without major thrombosis. Other predictors of survival were high-risk IPSET score and age >60 years. Conclusions: Romanian patients diagnosed with ET are generally younger than 60 years and are predominantly female. The occurrence of thrombotic events was influenced by gender, leukocyte count at diagnosis and JAK2V617F positivity. Survival was impacted by age, the presence of JAK2V617F mutation, hypertension, major thrombotic complications and IPSET score. Notably, these findings warrant careful interpretation and further confirmation in the setting of prospective studies.