Neocentromeres are newly formed chromosomal regions that can replace the function of traditional centromeres and are well documented in human clinical studies. However, their occurrence in neoplasia, including acute leukemia, appears to be rare. We analyzed complex karyotypes in bone marrow cells from 113 patients with acute myeloid leukemia and one patient with acute lymphoblastic leukemia using centromeric/multicentromeric fluorescence in situ hybridization and identified four cases (3.5%) with derivative chromosomes exhibiting newly formed constrictions. Three of these patients had secondary leukemia following preexisting hematological disorders, suggesting a potential role for neocentromeres in disease progression. In all four cases, neocentromeres were detected on derivative chromosome 11. To our knowledge, this is the first report of neocentromeres derived from this chromosome in acute leukemia. All four patients in our study died; however, all exhibited complex karyotypes, which are independently associated with poor prognosis and an aggressive disease course. Neocentromeres are a rare but potentially important source of genomic instability in malignant diseases. Generally, the formation of a new constriction allows mitotic rescue of acentric chromosomes, preventing their loss. An increase in genomic alterations in tumor cells predicts a more aggressive disease course and adverse outcomes. Due to limited data, the prognostic significance of neocentromeres remains unclear. Further rigorous investigation is required to deepen our understanding of the mechanisms underlying neocentromere formation and their implications in cancer.

The addition of gemtuzumab ozogamicin (GO) to intensive chemotherapy (IC) has become a mainstay in treating patients with core binding factor acute myeloid leukemia (CBF-AML). However, evidence for the efficacy of GO in this particular subgroup is primarily based on meta-analytic data from different trials conducted more than a decade ago. In this registry-based study, we evaluated the impact of adding GO to IC in 265 CBF-AML patients from the SAL, AMLCG, and CELL cooperative study groups. Patients receiving GO had a 2-year overall survival of 90% compared with 80% in those without GO (hazard ratio [HR] 0.45, 95% confidence interval [CI] 0.21–0.95, P = 0.036) and a 2-year event-free survival of 51% versus 36% (HR 0.69, 95% CI 0.48–0.99, P = 0.046). While complete remission rates in GO vs. non-GO patients were comparable (89% vs. 90%, P = 0.81), more GO patients achieved measurable residual disease-negative remission (77% vs. 49%, P < 0.001), resulting in numerically reduced cumulative incidence of relapse (HR 0.67, 95% CI 0.43–1.02, P = 0.06). Despite delayed platelet recovery, high-grade toxicities were not increased in GO-treated patients. These findings support the integration of GO into treatment protocols for IC-eligible patients with CBF-AML.

BACKGROUND:Venetoclax (VEN) combined with azacitidine (AZA) has become the standard treatment for patients (pts) with newly diagnosed (ND) acute myeloid leukemia (AML) who are unfit for intensive chemotherapy. This study reports on a real-world cohort of 163 patients with ND AML treated with VEN/AZA across 7 hospitals in the Czech Republic. PATIENTS AND METHODS:The median age at diagnoses was 72 (39-98) years. Standard VEN/AZA (7 + 28; ie, AZA 75mg/m2 for 7 days + VEN for 28 days) was used in 89% of patients (145/163), with 73% (119/163) receiving a reduced dose of venetoclax (100 mg or 200 mg) due to concomitant azole antifungal prophylaxis. Treatment was considered palliative or nonintensive (including potential allogeneic transplant candidates) based on the clinician's decision. RESULTS:Among 150 evaluable patients, 71% (107/150) achieved composite complete remission (CCR = CR + CRi), with 28% (32/115) achieving MRD negativity. Median overall survival (OS) was 5 months for the palliative cohort and 21 months for the nonintensive cohort. Day 30 and day 60 mortality rate were 7% and 13%, respectively. Allogeneic transplantation (HSCT) was performed in 15% (25/163) of pts, with the median OS not reached in this group. Mutations in NPM1, and IDH1/2 were associated with improved survival, while FLT3, ASXL1, TP53, deletion/monosomy 7, and complex karyotype were linked to poorer outcomes. CONCLUSION:Data suggest that TP53 patients benefit from treatment if they achieve CR within the first 2 cycles. Discontinuing VEN/AZA in low-risk MRD-negative patients does not appear to worsen overall survival, though long-term outcomes remain to be seen.

In the European LeukemiaNet (ELN) 2022 recommendations, myelodysplasia-related (MR) gene mutations were classified as a novel adverse prognostic category for intensively treated acute myeloid leukemia (AML). To assess the prognostic impact of individual MR genes within the ELN, clinical, cytogenetic, and molecular data from 4,978 intensively treated AML patients were analyzed. Remission rates and survival outcomes were evaluated. For analyses in context of ELN2022 classification, patients carrying an MR mutation were excluded from the adverse group and analyzed separately; those with co-occurring favorable or intermediate features remained in their respective groups. Overall, 1698 patients (34.1%) harbored at least one MR mutation. Lower complete remission rates were observed in MR-mutated cases (65.7% vs 77.7%; p < 0.001) along with shorter event-free (HR 1.45; p < 0.001), relapse-free (HR 1.33; p < 0.001), and overall survival (HR 1.45; p < 0.001) were recorded. Gene-specific prognostic patterns emerged: ASXL1, RUNX1, SF3B1, and U2AF1 mutations associated with adverse risk-like outcomes; SRSF2 and STAG2 aligned with intermediate-risk; BCOR, EZH2, and ZRSR2 did not differ significantly from intermediate or adverse risk. These findings from a large cooperative cohort highlight prognostic heterogeneity among MR mutations and suggest that SRSF2 and STAG2 mutations are associated with less adverse risk patterns, comparable to intermediate-risk.

Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease—myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.

COVID-19 significantly impairs survival rates among hematological patients when compared to the general population. Our prospective multicentre project analyzed early administration of anti-SARS-CoV-2 spike protein neutralizing monoclonal antibodies (NmAbs) - bamlanivimab (72%) and casirivimab/imdevimab (28%) - efficacy among hematological patients with early-stage COVID-19. Mortality rate was compared to a control cohort of 575 SARS-CoV-2 positive hematological patients untreated with any specific anti-COVID-19 therapy. 88 hematological patients with lymphomas, acute leukemias, and myeloma as their most frequent underlying diagnoses (72%) were evaluated with a 97 days median follow-up after NmAb administration. One third of patients (32%) were treated with an anti-CD20 monoclonal antibody before COVID-19 diagnosis. Median time between first COVID-19 symptom and NmAb administration was 2 days. When administering NmAb, 29%, 57%, 11%, 2%, and 1% of our patients had asymptomatic, mild, moderate, severe, and critical degrees of COVID-19, respectively. 80% of baseline asymptomatic patients remained asymptomatic following NmAb administration. Median duration of COVID-19 symptoms after NmAb administration was 2.5 days. Progression to severe/critical COVID-19 occurred among a total of 17% (15/88) of our cases and numerically higher with bamlanivimab versus casirivimab/imdevimab (21% vs. 8%; p = 0.215), and myelomas (29%), lymphomas (17%) and acute leukemias (18%), respectively. During final follow-up, nine deaths (10%) were recorded - all after bamlanivimab (p = 0.056) with 8% attributed to COVID-19. Regarding "remdesivir/convalescent plasma naive" patients, COVID-19 mortality rates were significantly lower in our NmAbs treated cohort compared to the control cohort of untreated SARS-CoV-2 positive hematological patients (6% vs. 16%, p = 0.020), respectively. Our study validated the safety and efficacy of NmAbs early use among hematological patients with newly diagnosed early-stage COVID-19 in terms of alleviating infection course and decreasing mortality. Results confirmed a more positive effect of a casirivimab/imdevimab combination versus bamlanivimab monotherapy.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with large heterogeneity at the clinical and molecular levels. As diagnostic procedures shift from bone marrow biopsies towards less invasive techniques, circulating small noncoding RNAs (sncRNAs) have become of particular interest as potential novel noninvasive biomarkers of the disease. We aimed to characterize the expression profiles of circulating sncRNAs of MDS patients and to search for specific RNAs applicable as potential biomarkers. We performed small RNA-seq in paired samples of total plasma and plasma-derived extracellular vesicles (EVs) obtained from 42 patients and 17 healthy controls and analyzed the data with respect to the stage of the disease, patient survival, response to azacitidine, mutational status, and RNA editing. Significantly higher amounts of RNA material and a striking imbalance in RNA content between plasma and EVs (more than 400 significantly deregulated sncRNAs) were found in MDS patients compared to healthy controls. Moreover, the RNA content of EV cargo was more homogeneous than that of total plasma, and different RNAs were deregulated in these two types of material. Differential expression analyses identified that many hematopoiesis-related miRNAs (e.g., miR-34a, miR-125a, and miR-150) were significantly increased in MDS and that miRNAs clustered on 14q32 were specifically increased in early MDS. Only low numbers of circulating sncRNAs were significantly associated with somatic mutations in the SF3B1 or DNMT3A genes. Survival analysis defined a signature of four sncRNAs (miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p measured in EVs) as the most significantly associated with overall survival (HR = 5.866, p < 0.001). In total plasma, we identified five circulating miRNAs (miR-423-5p, miR-126-3p, miR-151a-3p, miR-125a-5p, and miR-199a-3p) whose combined expression levels could predict the response to azacitidine treatment. In conclusion, our data demonstrate that circulating sncRNAs show specific patterns in MDS and that their expression changes during disease progression, providing a rationale for the potential clinical usefulness of circulating sncRNAs in MDS prognosis. However, monitoring sncRNA levels in total plasma or in the EV fraction does not reflect one another, instead, they seem to represent distinctive snapshots of the disease and the data should be interpreted circumspectly with respect to the type of material analyzed.

BACKGROUND:We aimed to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with myelodysplastic syndrome (MDS).METHODS:Targeted enrichment resequencing of 84 DNA repair genes was initially performed on a screening cohort of MDS patients. Real-time polymerase chain reaction was used for genotyping selected SNPs in the validation cohort of patients.RESULTS:A heterozygous frameshift mutation in the XRCC2 gene was identified. It leads to the formation of a truncated non-functional protein and decreased XRCC2 expression level. Decreased expression levels of all DNA repair genes functionally connected with mutated XRCC2 were also present. Moreover, a synonymous substitution in the PRKDC gene and 2 missense mutations in the SMUG1 and XRCC1 genes were also found. In the screening cohort, 6 candidate SNPs were associated with the tendency to develop MDS: rs4135113 (TDG, p = 0.03), rs12917 (MGMT, p = 0.003), rs2230641 (CCNH, p = 0.01), rs2228529 and rs2228526 (ERCC6, p = 0.04 and p = 0.03), and rs1799977 (MLH1, p = 0.04). In the validation cohort, only a polymorphism in MLH1 was significantly associated with development of MDS in patients with poor cytogenetics (p = 0.0004).CONCLUSION:Our study demonstrates that genetic variants are present in DNA repair genes of MDS patients and may be associated with susceptibility to MDS.

Anémie, definovaná jako pokles koncentrace hemoglobinu pod dolní hranici normálních hodnot, je projevem mnoha chorobných stavů a pro adekvátní terapii pacienta je vždy nutné diferenciálně diagnosticky upřesnit její příčinu.Článek využívá morfologického dělení anemií na makrocytární, normocytární a mikrocytární a jako zvláštní skupinu vyčleňuje anémie se zvýšeným počtem retikulocytů v periferní

Anemia, defined as hemoglobin level under lower normal limit, is a symptom of different pathologic conditions and the accurate differential diagnosis is necessary to determine the cause of anemia. The article uses the morphological classification of anemia to distinguish macrocytic, normocytic and microcytic types of anemias and divides anemias with increased amount of peripheral blood reticulocytes as a special group. It describes commonly known clinical units as iron deficiency anemia or anemia of chronic disease, as so as rare clinical units, which are always need to think about in a differential diagnosis of an anemic patient. There is an increasing incidence of rare blood disorders due to introduction of molecular genetics methods into diagnostics, prolonged overall survival of patients and increasing migration from areas with endemic occurrence of these diseases. Etiology, basic pathophysiological mechanisms, main clinical features together with important diagnostic examinations are described by each clinical unit. Due to the differential diagnostic focus of the article only basic knowledge about therapy is mentioned. The authors are members of the IHBT Center for Rare Disorders of Hematopoiesis, which is focused mainly on congenital and acquired disorders of red blood cell. Key words: anemia - differential diagnosis - enzymopathies - hemoglobinopathies - iron - macrocytosis - microcytosis - rare blood disorders - reticulocytosis.

A model of coupled limit cycle oscillators is developed and analyzed to discover and map out the system’s locking behavior. This sixth order model is motivated by the physical problem of a pair of closely spaced doubly clamped, thin silicon beams, coupled to each other through electrostatic fringing fields. The beams are assumed to be detuned with respect to each other. The beams are optically thin and are situated above a thick silicon substrate. When illuminated with continuous laser light a cavity interferometer is formed. Coupling of this optical interference with thermal stresses creates an inherent feedback loop that can drive the beams into limit cycle oscillation. Numerical analysis is used to study the range of coupling strengths and detunings over which 1:1, and other integer frequency ratio locking can be obtained. Results show that 1:1 locking can occur over a broad range of detuning even at relatively low levels of coupling. For coupling strengths just above the threshold for locking, both locked and drift states can exist, depending on the initial conditions. Locking at 2:1, 3:1, 3:2 and 5:2 frequency ratios are observed for detunings that are close but not exactly equal to these integer ratios.

TP53 mutations are frequently detected in patients with higher-risk myelodysplastic syndromes (MDS); however, the clinical impact of these mutations on the disease course of patients with lower-risk MDS is unclear. In this study of 154 lower-risk MDS patients, TP53 mutations were identified in 13% of patients, with prevalence in patients with del(5q) (23.6%) compared to non-del(5q) (3.8%). Two-thirds of the mutations were detected at the time of diagnosis, and one-third were detected during the course of the disease. Multivariate analysis demonstrated that a TP53 mutation was the strongest independent prognostic factor for overall survival (OS) (HR: 4.39) and progression-free survival (PFS) (HR: 3.74). Evaluation of OS determined a TP53 variant allele frequency (VAF) threshold of 6% as an optimal cut-off for patient stratification. The median OS was 43.5 months in patients with mutations detected at the time of diagnosis and a mutational burden of > 6% VAF compared to 138 months (HR 12.2; p = 0.003) in patients without mutations; similarly, the median PFS was 20.2 months versus 116.6 months (HR 79.5; p < 0.0001). In contrast, patients with a mutational burden of < 6% VAF were stable for long periods without progression and had no significant impact on PFS or OS. Additionally, we found a high correlation in the mutational data from cells of the peripheral blood and those of the bone marrow, indicating that peripheral blood is a reliable source for mutation monitoring. Our results indicate that the clinical impact of TP53 mutations in lower-risk MDS patients depends on the level of mutational burden.

Azacitidine (AZA) is a hypomethylating drug used to treat disorders associated with myelodysplasia and related neoplasms. Approximately 50 % of patients do not respond to AZA and have very poor outcomes. There is thus great interest in identifying predictive biomarkers for AZA responsiveness. We searched for specific genes whose expression level was associated with response status. Using microarrays, we analyzed gene expression patterns in bone marrow CD34+ cells in serial samples from 32 patients with myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia with myelodysplasia-related changes before and during the AZA therapy. At baseline, a comparison of the responders and non-responders showed 52 differentially expressed genes (P < 0.01). Functional annotation of the deregulated genes revealed categories primarily related to ribosomes and pathways associated with proliferation. The expression level of RPL28 correlated with overall survival. We identified altered expression in 167 genes in responders, 26 genes in non-responders with stable disease, and 13 genes in non-responders with disease progression using paired t test of expression levels in patients before and during treatment. Our data indicate that AZA treatment failure is associated with the up-regulation of ribosomal genes/pathways that are likely related to intensive proteosynthesis in proliferative/neoplastic cells of non-responders.

Background: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests that defects in DNA repair mechanisms. We monitored DNA repair pathways in MDS and their alterations during disease progression. Methods: Expression profiling of DNA repair genes was performed on CD34+ cells, and paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 was done on histology samples. Results: RAD51 and XRCC2 showed differential expression between low-risk and high-risk MDS (P<.0001), whereas RPA3 was generally decreased among the entire cohort (FC=-2.65, P<.0001). We demonstrated that RAD51 and XRCC2 expression gradually decreased during the progression of MDS. Down-regulation of XRCC2 and RAD51 expression was connected with abnormalities on chromosome 7 (P=.0858, P=.0457). Immunohistochemical staining revealed the presence of RAD51 only in the cytoplasm in low-risk MDS, while in both the cytoplasm and nucleus in high-risk MDS. The multivariate analysis identified RAD51 expression level (HR 0.49; P=.01) as significant prognostic factor for overall survival of patients with MDS. Conclusions: Our study demonstrates that the expression of DNA repair factors, primarily RAD51 and XRCC2, is deregulated in patients with MDS and presents a specific pattern with respect to prognostic categories.

Abstract Background and Aims Recent studies have demonstrated that in most cases of myelodysplastic syndrome (MDS) at least one mutation has been detected, suggesting that abnormal DNA repair may represent both cause and consequence of malignant transformation. In this study we investigated a possible role of different alterations in DNA repair genes in pathogenesis of MDS. Methods Gene expression of CD34+ cells was measured by RT² Profiler PCR Arrays (Qiagen). Polymorphisms and mutations were studied by targeting next generation sequencing (SeqCap EZ System, NimbleGen). Expression analysis of 84 DNA repair genes was performed in 18 MDS patients and subsequent analysis of selected genes was performed on a cohort of 80 patients. The enrichment resequencing of 84 genes was done in 16 patients. Results Five differentially expressed genes between CD34+ cells of patient and control samples were identified (p<0.05). The increased expression was detected in MPG and XPC genes and decreased expression in RAD51, RPA3 and XRCC2 genes. RAD51 gene showed significantly higher expression in the patients with low-risk MDS forms (RA, RARS, del5q) compared to control group (p=0.0005) and to contrast down-regulated expression was detected (p=0.0002) in high-risk MDS patients (RAEB-1 and -2, AML with myelodysplasia). The group of patients with RCMD showed the average expression at the level of the control group. The expression profile shown a gravity-related decreasing trend after dividing the patients according to IPSS-R, IPSS-R cytogenetic groups and blasts count. Expression profile of XRCC2 gene has similar characteristics as RAD51 (p<0.0001). The expression of RPA3 gene was generally decreased (p<0.0001) with a decreasing trend depending on the disease severity according to MDS forms, IPSS-R and cytogenetics. The RAD51, XRCC2 and RPA-3 genes are related to homologous recombination mechanism, with XRCC2 and RPA3 as a supporting factors for RAD51. The survival curves for all the three genes shown significant differences between groups with over-, intermediate- and down-expressed gene (p=0.0001 for RAD51; 0.0022 for XRCC-2 and 0.0054 for RPA3). MPG gene expression was up-regulated by all MDS types with no significant difference between each other (p=0.0015). Deeper testing of XPC gene expression demonstrated the up-regulation only in low-risk MDS group (p=0.0084). Gene-based analysis showed seven candidate SNPs significantly associated with the disease susceptibility using the HapMAP-CEU population from NCBI PubMed database as control group. With the presence of MDS, these polymorphisms are significantly connected: rs4135113 (p=0.03), rs12917 (p=0.003), rs2230641 (p=0.01), rs2228529 and rs2228526 (p=0.04, respectively p=0.03) and rs1799977 (p=0.04). Within the distribution of tested patient groups according to cytogenetics, we observed significant increase of dependency of these polymorphisms (OR 4.1-9.7, p<0.0001) and the presence of MDS in the group of patients with very-high risk cytogenetics. Conclusions DNA repair mechanisms are responsible for correcting DNA damage and preserving genomic integrity. Our study demonstrates, that altered expression of the repair genes in CD34+ cells may be responsible for the formation and accumulation of mutations in hematological malignancies. Furthermore, we have identified genetic variants that might contribute to the pathogenesis of MDS by modifying individual risk for the disease. Supported by grant (NT/13899, NT/14377, and NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.