Percentage Of Blast Cells Research Articles

Factors Associated with Achieving Hematological Response in CML (Chronic Myeloid Leukemia) Patient

Background: The goal of CML therapy is to achieve complete hematological response first, then cytogeneticresponse andcomplete molecular response at the very end. Achieving a complete hematological responseis the easiest and relatively inexpensive to detect. Several factors are thought to have an association withachieving hematological response. This study sought to determine the factors associated with achievinghematological response in CML patientsMethod: We conducted an observational retrospective cohort study of CML patients at Wahidin SudirohusodoHospital and its network on 2018-2019 period. Diagnosis was done by positive BMP (Bone Marrow Punctie)results for CML, then a monthly blood check until a hematological response was achieved.Results: There wer 39 research subjects, 19 male (48.7%) and 20 female (51.3%). No association foundbetween age, gender and achievement of hematological response. Type of therapy was significantlycorrelated with the achievement of hematological response, with the highest number found in Nilotinibtherapy compared to other group. (54,1%; p = 0.046) Subjects who achieve hematological response had alower percentage of blast cells. (6.1% vs. 7.9% ; p = 0.016)Conclusion: The use of Nilotinib therapy was significantly associated with a succesful achievementof hematological response. A lower percentage of blast cells was found in CML patients who achievedhematological response.

Gene coexpression network analysis revealed biomarkers correlated with blast cells and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy with a poorly understood pathogenesis, especially among patients with no known cytogenetic abnormalities. Furthermore, there is a lack of therapeutic gene targets and diagnostic biomarkers for the effective treatment of AML. The present study aimed to identify candidate biomarkers correlated with the clinical prognosis of patients with AML. Leukemic cells from 5 patients with AML exhibiting a normal karyotype, and hematopoietic cells from 5 healthy donors were processed for RNA sequencing (RNA-seq), and the obtained RNA expression profiles were subjected to weighted gene correlation network analysis. A novel group of genes (the red module) were identified to be significantly associated with AML, and this module contained a closely connected network with 147 nodes, which corresponded to 114 mRNAs. Analysis of the correlation between these mRNAs and blast cell percentage, overall survival (OS) and disease-free survival (DFS) using cases from The Cancer Genome Atlas (TCGA) database revealed that CSF3R, ALPL and LMTK2 were negatively associated with the percentage of blast cells, while high expression of these genes was associated with longer OS and DFS in patients with AML. The differential expression of these three genes between patients with AML and healthy control subjects was supported using the Genotype-Tissue Expression and TCGA databases and was further confirmed using reverse transcription-quantitative (RT-qPCR). These genes exhibited significantly lower expression in patients with AML compared with control subjects. The results indicated that CSF3R, ALPL and LMTK2 exhibit the potential to be prognostic biomarkers. However, the biological functions of these three candidate genes need to be assessed in further studies.

Analysis of RUNX1 Gene Mutation in Patients with Myelodysplastic Syndrome

To investigate the mutation of RUNX1 gene in patients with myelodysplastic syndrome (MDS) and its correlation with other gene mutations and some clinical parameters. The mutations of RUNX1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT in 170 patients with MDS were detected by direct and indirect sequencing of genomic DNA-PCR amplification products. The RUNX1 mutation was found in 23 patients (13.5 %, 23/170). Among the 170 patients, other most frequent mutation was TET2 (11.2%, 19/170), followed by mutations in DNMT3A (9.4%, 16/170), NPM1 (8.2%, 14/170), IDH2 (4.1%, 7/170)、FLT3-ITD (2.9%, 5/170), IDH1 (1.7%, 3/170) and c-KIT (0.58%, 1/170). The most common coexisting mutations were TET2 (5/23). The RUNX1-mutated group showed significantly higher leukocyte levels, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet counts in comparison with RUNX1 non-mutation group (P＜0.05). whereas there were no statistically significant difference in age, MDS subtype, karyotype and hemoglobin level between 2 groups (P＞0.05). Seventeen patients harboring RUNX1 mutations were followed up and almost 47.05% (8/17) of the patients progressed into acute myeloid leukemia (AML). The rates of transformation into AML in ASXL1-mutation group was significantly higher than that in ASXLL- non-mutation group (47.05% vs 11.7%) (P=0.001). The incidence of RUNX1 mutation is high in MDS patients. The RUNX1-mutated patients have higher leukocyte level, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet count.

Analysis of RUNX1 Gene Mutation in Patients with Myelodysplastic Syndrome

To investigate the mutation of RUNX1 gene in patients with myelodysplastic syndrome (MDS) and its correlation with other gene mutations and some clinical parameters. The mutations of RUNX1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT in 170 patients with MDS were detected by direct and indirect sequencing of genomic DNA-PCR amplification products. The RUNX1 mutation was found in 23 patients (13.5 %, 23/170). Among the 170 patients, other most frequent mutation was TET2 (11.2%, 19/170), followed by mutations in DNMT3A (9.4%, 16/170), NPM1 (8.2%, 14/170), IDH2 (4.1%, 7/170)、FLT3-ITD (2.9%, 5/170), IDH1 (1.7%, 3/170) and c-KIT (0.58%, 1/170). The most common coexisting mutations were TET2 (5/23). The RUNX1-mutated group showed significantly higher leukocyte levels, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet counts in comparison with RUNX1 non-mutation group (P＜0.05). whereas there were no statistically significant difference in age, MDS subtype, karyotype and hemoglobin level between 2 groups (P＞0.05). Seventeen patients harboring RUNX1 mutations were followed up and almost 47.05% (8/17) of the patients progressed into acute myeloid leukemia (AML). The rates of transformation into AML in ASXL1-mutation group was significantly higher than that in ASXLL- non-mutation group (47.05% vs 11.7%) (P=0.001). The incidence of RUNX1 mutation is high in MDS patients. The RUNX1-mutated patients have higher leukocyte level, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet count.

Evaluation of interleukin-35 and interleukin-10 in adult acute myeloid leukemia patients before and after induction chemotherapy

BACKGROUND: Acute myeloid leukemia (AML) is a clonal proliferation of hemopoietic cells. Interleukin-35 (IL-35) is a pro-inflammatory cytokine expressed in T regulatory (Treg) and B regulatory cells. IL-35 promotes the proliferation of AML blasts and lessens apoptosis. Hence, IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML. IL-10 is anti-inflammatory cytokines with immune-stimulatory activities and is formed by CD4 and CD8 T-cells and activated B-lymphocytes.OBJECTIVES: The aim of th study was to estimate the levels of IL-10 and IL-35 in the sera of patients with AML before and after chemotherapy induction and to correlate levels with blast cells percentage and other hematological parameters.PATIENTS, MATERIALS AND METHODS: This study was conducted on thirty newly diagnosed (ND), de novo adult AML patients, 15 males and 15 females with a age range between 19 and 75 years for a period from September 20, 2017, to March 15, 2018. It included 18 healthy (9 males and 9 females) individuals who were taken as a control group. Diagnosis of AML was established according to the morphology, cytochemistry and flow cytometry study of both peripheral blood and bone marrow aspiration as well as biopsy reports. ILs-35 and 10 levels were measured at diagnosis and after induction chemotherapy when achieving complete remission based on Cheson et al. definition.RESULTS: Serum IL-35 and IL-10 were significantly higher in ND AML patients than controls and were reduced after induction chemotherapy (P < 0.001). In patients with remission, IL-10 was significantly reduced compared with non-remission group, while the reduction in IL-35 level in remitted compared to nonremitted patients did not reach the level of significance (P > 0.001). No correlations were found between hemoglobin, platelet counts, white cell count, and blast percentage with IL-35 and 10 levels in de novo AML patients.CONCLUSIONS: Both ILs-35 and 10 levels were elevated in de novo AML patients and lowered following induction chemotherapy. Both ILs have no correlation with hemoglobin level, platelet count, white blood cell count, and blast percentage.

Involvement of RUNX1 Pathway Is a Common Event in the Leukemic Transformation of Chronic Myeloproliferative Neoplasms (MPNs)

Introduction. Runt-related transcription factor 1 (RUNX1) is a transcription factor critical for normal and leukemic hematopoiesis. Three main isoforms, RUNX1 i-1a, i-1b and i-1c, are produced through alternative splicing of RUNX1. Unlike i-1b and i-1c, i-1a has a Runt homology domain (RHD) but lacks the transactivation domain (TAD), and inhibits transcriptional activity of -1b by competing with higher affinity for target gene sequences. Overexpression of i-1a in cell lines inhibits terminal myeloid differentiation. The i-1a is expressed at high levels in patients (pts) with acute myeloid leukemia (AML). Mutations of RUNX1 are common in CMML and MDS, whereas incidence and clinical correlates of RUNX1 mutations in MPNs are poorly known. Aim: To analyze the spectrum and clinical correlates of RUNX1 genetic abnormalities (RUNX1-abn), and the expression of RUNX-1a isoform, in a large series of pts with MPNs. Methods. 686 MPN pts (50 PV, 165 ET, 154 overt-PMF, 143 prefibrotic-PMF, 74 PPV-MF and 100 PET-MF, diagnosed according to 2016 WHO) were included in the study; of these, 625 were analyzed in chronic phase (CP) and 61 at leukemic transformation (LT; of a total of 106 LTs, 58%), including 29 cases with paired samples at diagnosis and LT. Established methods were used for JAK2, MPL and CALR genotyping. A high molecular risk (HMR) category was defined according to Vannucchi A, [Leukemia 2013]. Ion Torrent NGS platform was used to genotype all RUNX1 coding sequence. In 21/29 paired samples, a long reads genome sequencing by Oxford Nanopore technology (ONT) was performed. Copy number variation (CNV) analysis was performed with NanoGLADIATOR package [Magi A, Genome Biol 2019]. Differential expression of RUNX1 mRNA isoform1a and 1b was analyzed by qRT-PCR. Nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimates and log-rank test were used as appropriate. Results. Somatic RUNX1 mutations were identified in 31 pts (4.5%): 14/625 in CP (2.2%) and 17/61 in LT (27.9%; P&lt;.0001); 7/17 LT pts (41.1%) had two variants. 10/29 paired samples were RUNX1 mutated, in 8 of which mutations were acquired at LT. All mutations were heterozygous (median VAF 40%, range 0.4%-54%); 13 (34.2%) were frameshift, 5 (13.2%) nonsense and 20 (52.6%) missense. Most mutations clustered in RHD (26/38, 68.4%) and TAD (9/38, 23.7%). CNV involving RUNX1 was detected by ONT in 4/21 (19%) paired samples, including 2 gain and 2 loss, all acquired at LT; 2 pts had concurrent RUNX1 mutation. Overall, RUNX1abn, inclusive of mutations and CNV, occurred in 19/61 (31.1%) patients at LT. RUNX1-abn were more common in MPL (12.5%) and TN (7.5%) pts compared with JAK2 (4.3%) and CALR (2.0%) mutated (P=0.04), and were enriched in HMR category (61.3% vs 29.4% vs pts without RUNX1-abn; P=.001). We also analyzed CNV in a total of 47 selected genes included in the RUNX1 pathway (KEGG Orthology K08367); we found CNV in &gt;1 of these genes in 16/21 (76%) pts at LT vs 2/16 (12.5%) of CP pts. After median follow-up of 4.9y (range 0.1-36.2y) 273 pts died (39.8%). 106 pts (15.4%) progressed to AML after median of 4.7y (0.1-36.2y) from diagnosis. Incidence of LT differed significantly among diseases: 3 pts in PV (5.9%), 17 in ET (10.1%), 39 in overt-PMF (23.5%), 12 in prefibrotic-PMF (8.3%), 11 in PPV-MF (14.1%) and 24 in PET-MF (22.6%). RUNX1-abn were detected, respectively, in 100%, 53.0%, 23.1%, 41.7%, 36.4% and 12.5% of cases at either CP and/or LT. Leukemia-free survival (LFS) was shorter in pts harboring RUNX1-abn in CP compared to RUNX1-WT pt (2.8y vs NR; P&lt;.0001)(Fig 1A). Expression levels of i-1a and -1b were measured in 34 CP and 24 LT pts. Isoform-1a was significantly elevated in LT pts (mean 7.51±10.1) compared to CP (1.12±1.75; P=.009) (Fig 1B; lines represent the i-1a level rise from CP to LT in 4 paired samples). The i-1a expression levels were irrespective of RUNX1 mutated (mean 7.6±11.9) or unmutated (mean 9.2±6.3) status in LT pts (P=0.8). No difference of i-1b levels in CP (mean 0.9±0.7) versus LP (mean 1.6±1.4; P=0.08) was observed. The expression of i-1a was not dependent on gender, age, leukocyte or platelet count, hemoglobin and percentage of blast cells. Conclusions. These findings indicate that most MPN pts with LT have involvement of RUNX1 pathway through different mechanisms (mutations, CNV, altered isoform expression, genomic abnormalities of RUNX1 target genes), suggesting that a "RUNX1pathy" may represent a common event during leukemogenesis in MPNs. Figure 1 Disclosures Vannucchi: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Combination of the MEK Inhibitor Trametinib and Pyrvinium Pamoate Efficiently Targets RAS Pathway-Mutated Acute Myeloid Leukemia in Preclinical Models

While acute myeloid leukemia (AML) is still associated with a low cure rate, recent advances in understanding its molecular complexity have significantly improved therapy for subgroups of patients, including those harboring FLT3, IDH1 or IDH2 mutations1. However, more than half of AML cases still lack a druggable oncogenic target. Human cancers frequently harbor mutations in RAS oncogene family members, including NRAS, KRAS and HRAS, which drive oncogenesis by augmenting cellular proliferation and survival. These are small protein GTPases, regulated by a switch between active GTP-linked and inactive GDP-bound states governed by a complex network of guanine exchange factors (GEFs, favoring RAS-GTP) and GTPase activating factors (GAPs, favoring RAS-GDP). RAS activation - due to either extrinsic recruitment by transmembrane tyrosine kinase receptors or intrinsic mutations - propagates through the downstream RAF/MEK/ERK and PI3K/AKT signaling pathways. Besides RAS-activating mutations conferring independence from physiological regulators, human cancers harbor mutations in other RAS network genes such as NF1 (encoding neurofibromin, a RAS GAP), BRAF or PTPN11 (encoding the SHP2 tyrosine phosphatase involved in RAS activation). Somatic alterations of RAS pathway genes, notably NRAS, KRAS, PTPN11 (missense mutations) and NF1 (mutations and deletions), are reported in up to 20% of AML cases2. Generally arising as late driver events, RAS pathway mutations participate in leukemogenesis through mitogen activated protein kinase (MAPK) activation. The anti-tumor activity of MEK inhibitors in Nras-mutated AML in mice and in some NRAS or KRAS-mutated AML patients suggests that deregulated RAS pathway signaling may represent a bona fide therapeutic target. However, strategies to inhibit RAS - indirectly in most cases - have been hampered by signaling feedback, redundancy and tumor heterogeneity 3. We identified 127 cases of AML with unmet therapeutic need within a cohort from which we excluded those with European leukemia network (ELN) favorable prognosis or FLT3-ITD mutations. Targeted next-generation sequencing revealed RAS pathway alterations in 50 patients (39.3%) and NF1 mutations and deletions, mostly large cytogenetically detected deletions, in 17 (14.8%). NRAS, KRAS, PTPN11, CBL and BRAF variants were detected in 13 (10.4%), 10 (7.9%), 9 (7.2%), 5 (3.9%) and 2 (1.6%) cases, respectively. Mutations in RAF1, RASA1, SOS1 and MAP2K2 were observed in a single case each. RAS pathway alterations appeared in the putative main leukemic clone as well as in subclones inferred from variant allele frequencies. Concurrent RAS pathway mutations were observed in nine cases. Among 79 patients homogeneously treated with intensive induction chemotherapy, RAS pathway alterations correlated with higher clinical proliferation markers (elevated white blood cell count, blast cell percentage and LDH levels) and reduced survival probability, particularly within the ELN intermediate-risk subgroup. We established robust models of RAS/MAPK activation through genetic NF1 disruption or expression of NRASG12D or PTPN11D61Y in growth factor (GF)-dependent cell lines. We assessed oncogenic addiction to the RAS pathway in these cells through GF-independence, increased RAS activity, faster propagation in immunocompromised mice and an exquisite sensitivity to pharmacological MEK inhibition in vitro and in vivo. High-content pharmacological screens with FDA-approved molecules identified pyrvinium pamoate, an anti-helminthic agent, as preferentially active in RAS-activated cells. This compound significantly impaired cell viability and colony formation in primary AML samples with RAS pathway alterations. Moreover, the combination of trametinib and pyrvinium pamoate demonstrated synergy in cell line models and even primary samples. While pyrvinium pamoate strongly inhibited mitochondrial respiration and induced metabolic reprograming towards increased glycolysis, trametinib impaired glycolysis and mitochondrial respiratory capacity, suggesting a mechanistic basis for the synergy observed. These data highlight the translational opportunity in developing pyrvinium pamoate for RAS pathway mutated AML.

Cooperation of Dnmt3a R878H with Nras G12D promotes leukemogenesis in knock-in mice: a pilot study

BackgroundDNMT3A R882H, a frequent mutation in acute myeloid leukemia (AML), plays a critical role in malignant hematopoiesis. Recent findings suggest that DNMT3A mutant acts as a founder mutation and requires additional genetic events to induce full-blown AML. Here, we investigated the cooperation of mutant DNMT3A and NRAS in leukemogenesis by generating a double knock-in (DKI) mouse model harboring both Dnmt3a R878H and Nras G12D mutations.MethodsDKI mice with both Dnmt3a R878H and Nras G12D mutations were generated by crossing Dnmt3a R878H knock-in (KI) mice and Nras G12D KI mice. Routine blood test, flow cytometry analysis and morphological analysis were performed to determine disease phenotype. RNA-sequencing (RNA-seq), RT-PCR and Western blot were carried out to reveal the molecular mechanism.ResultsThe DKI mice developed a more aggressive AML with a significantly shortened lifespan and higher percentage of blast cells compared with KI mice expressing Dnmt3a or Nras mutation alone. RNA-seq analysis showed that Dnmt3a and Nras mutations collaboratively caused abnormal expression of a series of genes related to differentiation arrest and growth advantage. Myc transcription factor and its target genes related to proliferation and apoptosis were up-regulated, thus contributing to promote the process of leukemogenesis.ConclusionThis study showed that cooperation of DNMT3A mutation and NRAS mutation could promote the onset of AML by synergistically disturbing the transcriptional profiling with Myc pathway involvement in DKI mice.

Analysis of mutations of 14 genes among 87 patients with myelodysplastic syndrome

To explore the correlation of genetic mutations and clinical features of myelodysplastic syndromes (MDS) with scores of Revised International Prognostic Scoring System (IPSS-R). Eighty-seven patients with de novo MDS were enrolled. Mutations of MDS-related genes and clinical features were used to determine the incidence and subtype of mutations. Clinical features and IPSS-R scores of the patients with high frequency mutations involving TET2, TP53, ASXL1, RUNX1 and SF3B1 genes were compared. Fifty-four patients (62.1%) harbored at least one point mutation. The incidences of various mutations were significantly different, with the incidence of MDS-EB-2 being 100% and MDS-SLD being only 38.9%. Compared with the wild types, patients harboring mutations had higher lactate dehydrogenase, higher β2 microglobulin, higher percentage of bone marrow blast cells and lower hemoglobin levels (P=0.027, <0.01, <0.01, 0.046, respectively). The IPSS-R scores of MDS patients with mutations were significantly higher than the wild types (P<0.01). The IPSS-R scores of the TP53 mutation groups were 7.82±1.83, which was significantly higher than the control group (3.77±1.66, P<0.01). No difference was found between the IPSS-R between patients carrying TET2, ASXL1, RUNX1, and SF3B1 mutations or the wild types (P>0.05). Genetic mutations are commonly found in MDS. MDS patients with mutations have unique clinical laboratory characteristics. Although the prognostic value of most genes is controversial, TP53 is an definite indicator of poor prognosis.

Prognostic Value of CD45 Surface Antigen in Children with Acute Lymphoblastic Leukemia Treated according to Total XV Protocol

AbstractBackground: Acute Lymphoblastic Leukemia (ALL) is a malignant disease of the bone marrow in which early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow. Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. CD45 cell surface antigen is a transmembrane protein with tyrosine phosphatase activity, expressed by all nucleated cells of hematopoietic origin, except erythrocytes and platelets.Aim of Study: To assess the prognostic value of CD45 surface antigen in children with acute lymphoblastic leukemia who treated according to TOTAL XV protocol.Subjects and Methods: This study was conducted on 25 patients with acute lymphoblastic leukemia who treated according to Total XV protocol. CD45 expression was meas-ured by flowcytometry and cut-off value for CD45 expression is set on 90% to distinguish a CD45-high from a CD45-low group.Results: There was significant correlation between CD45 expression and age. There was no statistically significant differences between low and high CD45 groups as regard initial total leucocytic count, bone marrow blast cells percent-age, leukemic phenotype, DNA index, t (9, 22) or CNS status. There was statistically significant difference between the two groups regarding early treatment response, risk stratification, incidence of relapse and event free survival.Conclusion: Measuring the intensity of expression of CD45 surface antigen has a prognostic value in childhood acute lymphoblastic leukemia. So consideration of CD45 expression may serve as an additional stratification tool.Recommendations: Multicenter study on large number of patients with follow-up for longer duration is recommended to prove the prognostic value of CD45 expression in childhood acute lymphoblastic leukemia.

Significance of apoptosis and autophagy of leukemic blasts for the outcomes of acute myeloid leukemia patients

Cytostatic treatment induces apoptosis or other types of cell death like autophagy, necrosis, mitotic catastrophe, etc. Autophagy can play a role in the drug resistance of neoplastic cells, allowing the survival of blast cells under stressful conditions, such as the use of cytostatics. Studies on apoptosis and autophagy 12-24 h after the start of treatment have not been conducted until now. The study aimed to investigate the predictive and prognostic significance of autophagy and apoptosis in patients with acute myeloid leukemia (AML). The study included 38 patients. Blood was collected before and 12-24 h after the start of treatment, since at that time point, the appropriate blast cell count was still available. Autophagy was measured with the expression of the ATG5, MAP1L3, LC3-I, and LC3-II proteins. The percentage of mononuclear cells in early and late apoptosis was evaluated with flow cytometry, using the annexin V and propidium iodide (PI) binding assay. The percentage of apoptotic blast cells before treatment was not associated with the response. However, in the remission group, the overall percentage of apoptotic cells measured 12-24 h after the start of treatment was higher than in non-remission patients, which was statistically significant. In neither group we found any difference in the level of autophagy before and 12-24 h after the start of treatment. Nevertheless, we observed an increasing tendency of the MAP1LC3 protein expression (not statistically significant) in the remission group 12-24 h after the start of treatment. Patients with a higher percentage of blast cells in apoptosis and with a higher expression of MAP1LC3 protein measured 12-24 h after the start of the therapy had longer overall survival (OS). A higher percentage of apoptotic as well as autophagic blast cells measured 12-24 h after the start of the chemotherapy is an independent factor associated with better outcomes.

PB2091 HOST INFLAMMATORY FACTORS MIGHT PREDICT LEUKEMIC TRANSFORMATION AND OVERALL SURVIVAL IN CHRONIC MYELOMONOCYTIC LEUKEMIA

Background:Chronic myelomonocytic leukemia (CMML) is a clonal myeloid malignancy with a variable clinical course and 15‐30% risk of progression to acute myeloid leukemia. Prognosis can vary according to various pretreatment patients’ factors. Scoring systems have been developed based on clinical and cytogenetic parameters1. Among patient related factors, host inflammatory anti‐neoplastic factors, e.g. neutrophils, monocytes, platelets, can contribute significantly to disease progression by promoting cancer cell proliferation, evasion of immune‐surveillance, etc., thus playing key roles in patients’ survival.Aims:The aim of the study was to evaluate the clinical and laboratory features at the initial diagnosis and their impact on the survival outcome of the investigated cohort.MethodsWe retrospectively collected clinical and hematologic data for 75 consecutive patients with CMML diagnosed at the National Hematology Hospital, Sofia in the period 2013‐2018. The diagnosis was defined according to WHO 2016 revised criteria. Demographic, clinical and laboratory data were recorded at the initial presentation. Overall survival (OS) was calculated as the time from diagnosis until death from any cause or last contact.Results:The patients’ cohort comprised of 49 males (65%) and 26 females (35%) at a mean age of 68,6 (ranging 26‐90). According to the FAB criteria the CMML cases with myelodysplastic features (WBC &lt; 13 G/l) were 28% (n = 21) vs CMML with myeloproliferative characteristics (WBC &gt; 13 G/l) 72% (n = 54). Progression to acute myeloid leukemia occurred in 9% of the cases for a 5‐year period of time and was significantly correlated with absolute neutrophil counts (ANC) (33% in pts with ANC &lt; 1,8 × 109/l vs 67% in pts with ANC ≥1,8 × 109/l; log rank p = 0,05), female gender (29% male vs 71% female pts; log rank p = 0,04) and splenomegaly (p = 0,03). No relevant differences were found in regard to hemoglobin levels, blast cell percentage, degree of myelofibrosis, number of dysplastic lineages, lactate dehydrogenase level or the therapeutic approach. We detected JAK2 V617F activated mutation in five (9,6%) of CMML patients, all younger than 70 years (p = 0,02), however JAK2 V617F mutational status did not correlate with clinical and laboratory parameters and also did not impact either leukemia free (LFS), nor overall survival (OS). Further analysis showed that in terms of OS the presence of constitutional symptoms, monocytes ≥3 × 109/l and platelets &lt;100 × 109/l were associated with adverse survival outcome. CMML cases with absolute monocyte count &lt;3 × 109/l had longer OS (82% vs 62,5% OS in pts with ≥3 × 109/l, mean 44 vs 16 months, respectively; log rank p = 0,03). Similarly, platelet count ≥100 × 109/l correlated with better outcome in the overall cohort (94% vs 64% OS in pts with thrombocytopenia, mean 42 vs 29 months, respectively; log rank p = 0,02) (Figure 1).Figure 1. Overall survival curves for patients with CMML according to the platelet count and absolute monocyte count at the initial diagnosis.Summary/Conclusion:Conclusion: In addition to some well established factors, e.g. gender and splenomegaly, our study provided data that pre‐treatment levels of some systemic inflammatory markers such as neutrophils, monocytes and platelets could be used in addition to predict leukemia transformation and shorter overall survival in CMML. Further research is needed to elucidate the underlying pathophysiological background for predictability of combined inflammatory biomarkers.image

Assessment of Bone Marrow Status in Different Hematological Diseases Using Diffusion Weighted Image (DWI)– and Apparent Diffusion Coefficient (ADC)–MRI Technique

Assessing the value of diffusion-weighted imaging (DWI) magnetic resonance imaging (MRI) and apparent diffusion coefficient (ADC) in normal bone marrow (BM) tissue as well as in benign and malignant hematological diseases in comparison with BM histopathologic report. A cohort cross-sectional study performed from October 2016 till December 2017 in Al-Imamian Al-Khadimain Medical City. Forty patients were enrolled and segregated equally into two main groups (benign and malignant hematological disorders with another 20 healthy volunteers (control group). Hematology data were recorded for all patients in addition to DWI-MRI and ADC mapping in comparison with bone marrow report for the group of malignant hematological disease in terms of blast cell percentage. Conventional MRI reveals abnormal signal intensity in 85% of the malignant cases with different pattern. While 95% of the benign and 100% of control group had normal signal intensity with P value (P = 0.001). Application of DWI-ADC on three groups of sample reveals a variable range of ADC values which is higher in malignant (225–875 × 10−6 mm2\s) than benign (275–600 × 10−6 mm2\s) and control cases (205–560 × 10−6 mm2\s) with a cut-off value of 550 × 10−6 mm2\s. There was a positive correlation between ADC values and blast % in bone marrow histopathological report with correlation coefficient of 0.75 and P value of 0.05. Both DWI-MRI and ADC are useful in BM assessment. The latter is a quantitative assay for the diffusion and can reflect a functional assessment in correlation with blast cell percentage.

Acute lymphoblastic leukemia segmentation using local pixel information

The severity of acute lymphoblastic leukemia depends on the percentages of blast cells (abnormal white blood cells) in bone marrow or peripheral blood. The manual microscopic examination of bone marrow is less accurate, time-consuming, and susceptible to errors, thus making it difficult for lab workers to accurately recognize the characteristics of blast cells. Researchers have adopted different computational methods to identify the nature of blast cells; however, these methods are incapable of accurately segmenting leukocyte cells due to some major disadvantages, such as lack of contrast between objects and background, sensitivity to gray-scale, sensitivity to noise in images, and large computational size. Therefore, it is indispensable to develop a new and improved technique for leukocyte cell segmentation. In the present research, an automatic leukocyte cell segmentation process was introduced that is based on machine learning approach and image processing technique. Further, the characteristics of blast cells were extracted using 4-moment statistical features and artificial neural networks (ANNs). It was found that the proposed method yielded a blasts cell segmentation accuracy of 97% under different lighting conditions.

The prognostic value of matrix metalloproteinase-7 and matrix metalloproteinase-15 in acute myeloid leukemia.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, are involved in a variety of physiological and pathological processes. We analyzed 11 data sets from Gene Expression Omnibus Database and found that MMP7 and MMP15 were highly expressed in multiple carcinomas. GSE13204 showed that MMP7 and MMP15 were overexpressed in acute myeloid leukemia (AML) patients. The Cancer Genome Atlas data set exhibited that high expression of MMP7 or MMP15 in bone marrow (BM) of AML patients predicted poor overall survival. The χ 2 test results indicated that high expression level of MMP7 and MMP15 were correlated with high-risk stratification and high BM blast cell percentage in AML patients. To confirm these findings, we performed reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and found that MMP7 and MMP15 were highly expressed in three AML cell lines. Further study showed that MMP7 and MMP15 were highly expressed both in BM and peripheral blood in collected AML samples compared with healthy individuals. Additionally, long noncoding RNA (lncRNA) microarray of BM samples of AML patients revealed that multiple lncRNAs were correlated with MMP7 and MMP15, suggesting that lncRNAs might be involved in the pathogenesis of AML via modulating MMPs. In conclusion, our study uncovers the potential roles of MMP7 and MMP15 in the prognosis of AML.

Prognostically significant bone marrow cellular content changes in diffuse large B-cell lymphoma

Introduction. Increasing of blast cell percentage in bone marrow of diffuse large B-cell lymphoma patients is a sign of unfavourable prognosis. We estimated the frequency of this phenomenon and made attempts to idenify minimal bone marrow involvement in DLBCL by flow cytometry.Materials and methods. Study has been done in 60 DLBCL patients. Diagnosis in all cases was done according to WHO (2008) criteria. Bone marrow study was done on smears (myelogram). Immunophenotypic study was done with a large panel of B-lineage monoclonal antibodies as well as kappa and lambda light chains to membrane immunoglobulins.Results. Increase in blast cell content in bone marrow was noted in 73.3 % of DLBCL patients. That group of patients had lower surviv al. We used the following methods for identification of monoclonal B-lymphocytes: monoclonality according to membrane light immu noglobuline chains within mature B-lymphocytes and aberrancy of immunophenotype. Levels of different B-cell antigen expression – CD20, CD21, CD22, CD24. It was identified B-lymphocytes with aberrant expression of СD21.Conclusion. The most informative methods of identification of minimal bone marrow involvement in DLBCL are estimation of clonality according to light chain expression of mature B-lymphocytes (CD45++CD20+CD5–), as well as assessement of aberrancy of CD21 expression of mature B-lymphocytes.

Deregulated Expression of Long Non-Coding RNA AC092580.4 in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a malignant clonal proliferative disease originating from hematopoietic stem cells, characterized by anemia, hemorrhage, infection and organ infiltration. Most of the patients had poor prognosis and the molecular mechanism of AML is not clear. Therefore, it is urgent to find new molecular markers and therapeutic targets for AML. Long noncoding RNAs (lncRNAs) are a class of RNAs, which are longer than 200nt and without the ability of encoding functional proteins [A. Fatica et al., 2014]. Accumulating studies have found that lncRNAs play an important role in the development of hematological tumors, especially in AML [Huang J et al., 2014; Xing C-y et al., 2015]. In order to investigate the mechanism of lncRNAs in AML, we screened the differentially expressed RNAs in 5 AML patients and 5 IDA controls by microarray (GSE103828), and lncRNA AC092580.4 was verified markedly low expressed in AML (fold change=15.25, P= 0.0002). Then, We further explored the relationship between the expression levels of AC092580.4 and the clinical characteristics and prognosis of AML.Methods: Bone marrow (BM) samples from patients with AML and iron deficiency anemia (IDA) were collected and qRT-PCR was performed to verify the expression levels of AC092580.4 in AML patients and controls. The Receiver Operating Characteristic (ROC) was established to analyze whether AC092580.4 could be used as a bio-marker for newly diagnosed AML patients. The clinical data, including age, gender, percentage of blast cells in BM, chromosome and gene abnormalities, risk stratification, white blood cell counts and overall survival were collected. The correlation between the expression levels of AC092580.4 and clinical characteristics were analyzed by SPSS22.0.Results: The expression levels of AC092580.4 in newly diagnosed AML (n=83) was significantly lower (Figure A) than IDA (n=24) (p < 0.0001). The ROC (Figure B) was established and the area under curve was 0.868 (p < 0.001, CI: 0.780 - 0.957), which indicated that AC092580.4 could be used as an effective index to distinguish the newly diagnosed AML from the controls. Clinical data were collected from 83 newly diagnosed AML patients, 67% was the mean value of the percentage of blast cells in BM . It was found that the expression levels of AC092580.4 were significantly different between the high (≥67%) and low(<67%) percentage of blast cells (p=0.001), high white blood cell group (WBC≥100×109/L) and non high white blood cell group (WBC<100×109/L) (p=0.021). There was no significant difference of the expression levels in the patients with favourable, intermediate and adverse according to Diagnosis and management of AML in adults 2017 ELN recommendations from an international expert panel (p=0.328). The overall survival rate (Figure C) analysis showed that AML patients with high expression levels of AC092580.4 have a better prognosis than low expression group (p=0.018), which indicated that AC092580.4 may be used as an effective index to predict prognosis. To explore the mechanism of AC092580.4 in AML, we obtained sequencing data of 151 adult AML patients from The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/abouttcga/). The correlation coefficient between AC092580.4 and coding gene was estimated by Pearson correlation method. The results showed that the correlation coefficient between GATA3 and AC092580.4 (r = 0.679, P <0.05) was the most significant one among 27338 transcripts (Figure D). Therefore, we hypothesize that AC092580.4 may play an important role in the pathogenesis of AML by interacting with GATA3. GATA3 is a transcription factor of GATA family. While promoting the development of Th2 cells, it can also inhibit the Th1 polarization of Th cells, and eventually lead to higher expression of Th2, which may lead to the occurrence of tumor.Conclusions: Our study sheds light on a novel lncRNA AC092580.4 in AML, and provides its possible functional mechanism preliminary. Whether it can be used as a new powerful therapeutic target for AML treatment remains to be further studied. DisclosuresNo relevant conflicts of interest to declare.

Clinical Features, Laboratory Characteristics, and Response to Therapy in Patients with Acute Undifferentiated Leukemia

Introduction: Acute undifferentiated leukemia (AUL), also referred to as ambiguous lineage leukemia, has been recognized as a unique entity by the WHO classification of myeloid neoplasms since 2008, and is considered a rare subtype. It is characterized by lacking expression of markers diagnostic of myeloid or lymphoid lineages, and encompasses the provisional entity of myeloid/natural killer (NK) leukemia, which also expresses CD56 and other immature T-cell markers. Small studies have reported that AUL is associated with a poor prognosis; however, cytogenetic and molecular characterization of this subgroup of patients is lacking, as are the long-term outcomes of induction therapy and stem cell transplantation (SCT). The goal of this study was to assess clinical and laboratory data of patients diagnosed with AUL at our institution, focusing on its unique characteristics and response to initial therapy.Methods: Parameters assessed at diagnosis included: initial blood counts, bone marrow (BM) blast cell percentage by morphology, specific phenotypic characteristics of BM blasts, molecular mutations and translocations, as well as cytogenetic abnormalities. Immunophenotyping of BM cells at diagnosis was performed using eight-color flow cytometry. Multi-parameter Cytognos acute orientation tube (ALOT) and the combination of the following antigens were used: CD34/CD117/CD14/CD56/CD36/CD123/HLA-DR/CD45/CD64/CD11c. Data were acquired with FACSCanto II flow cytometer and analyzed with Infinicyt software. Myeloperoxidase staining by fluorescence-activated cell sorting (FACS) was considered negative if <3% of blast population stained positive. Data on BM response to initial therapy, based on morphology, immunophenotyping and biopsy, as well as the 18-month outcome following the diagnosis of the first patient in this cohort were assessed.Results: One hundred and sixteen cases of acute myeloid leukemia were diagnosed at the Rambam Health Care Campus between 11/2016 and 5/2018; 16 (1.4%) of them were defined as AUL, since they did not qualify for myeloid, B- or T-cell lineage according to the WHO classification. CD56 positivity was demonstrated in 8 (50%) cases, 5 of which were also CD7 positive. Patient median age was 63 years, 56% were men. None of the patients was found positive for either NPM1 or FLT3 mutations. Likewise, no core binding leukemias were diagnosed in this cohort. Sixty six percent (10/15) of the BM samples demonstrated cytogenetic and/or FISH abnormalities, with 26% complex karyotype cases, and 20% chromosome 7 aberrancies. All but two patients were treated with AML-oriented therapies. None of the patients achieved remission with induction therapy regardless of whether it included a chemotherapy backbone (82% of patients) or a hypomethylating agent. Seven patients have undergone allogeneic SCT, 4 of whom are in remission to date. Two of the remaining transplanted patients died of progressive disease and one developed extramedullary relapse 6 months post-transplant. While 8/16 (50%) patients are alive at 18-month follow-up, only 4 of them (25%) are in remission. Notably, all of the latter patients are SCT recipients. Six out of 8 CD56-positive patients have died of progressive disease, and the remaining two have not achieved remission.Conclusions: The present study, while confirming the poor prognosis of AUL patients, also suggests that a certain subset of such individuals could benefit from an allogeneic SCT. The high percentage of complex karyotype abnormalities, the lack of NPM1 mutations, and the expression of T/NK cell immaturity markers could partly account for the established poor outcome. However, since other molecular abnormalities described in the AML mutational landscape may be involved in AUL initiation/progression, extensive molecular sequencing of this patient population is required. A better understanding of the unique characteristics of this leukemia subtype is also crucial for the design of potentially improved treatment strategies for AUL patients. DisclosuresNo relevant conflicts of interest to declare.

Loss of Heterozygosity and Possible Loss of Allele Amplification in Homozygous Short Tandem Repeats Loci in Two ALL Patients with Central Nervous System and Bone Marrow Relapses

Introduction: Loss of heterozygosity (LOH) was described for many forms of cancer including leukemia. LOH may contribute to oncogenesis through the deletion of tumor suppressor genes due to appropriate allelic loss. LOH in short tandem repeats loci (STR) has been reported for solid tumors and is widely used as malignant cells population marker. It should be noted that little is known yet about possible loss of one of certain homozygous alleles in tumour cells and STR analysis might be a promising approach to investigate this phenomenon. STR analysis is used as a routine test for chimerism monitoring after bone marrow transplantation. It could not also be excluded that recipient's chimerism estimation may be distorted in some cases due to LOH issues.The aim: To investigate possible allele loss in homozygous loci in tumour cells and to assess the contribution of LOH to the error in mixed chimerism estimation.Patients and Methods: This study includes analysis of STR DNA-profiles of two ALL patients after allogeneic stem cells transplantation. Patient 1 was 54 years old female with B-ALL, she underwent allogenic BM transplantation but after it she had four central nervous system (CNS) relapses and three bone marrow relapses. Patient 2 was 23 years old male with preT-ALL, two CNS relapses: first before transplantation and second after it. The DNAs were isolated from bone marrow samples of patients in remission before transplantation, from donors blood samples, from cerebrospinal fluid during neuro relapse and from bone marrow samples. The presence of blast cells in bone marrow samples and cerebrospinal fluid was confirmed morphologically and cytogenetically. STR-profiles were assessed by polymerase chain reaction with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software.Results: The STR profile analysis for Patient 1 showed complete recipient chimerism in cerebrospinal fluid with LOH in 10 from 15 heterozygous loci (Fig.1a,b) comparative to control sample of DNA (bone marrow before transplantation). The same LOH pattern was detected in archival DNA sample from first spinal relapse. Mixed chimerism in bone marrow samples estimated by STR analysis was equal to blast cell percentage assessed by cytogenetics. Loss of non-informative allele in D16S539 (Fig.1b) didn't distort chimerism estimation. However decreased levels of mixed chimerism were calculated in two homozygous loci (Fig.1a,c). We suspected that this decrease could be caused by the loss of one allele amplification in homozygous locus. Therefore, we proposed the mathematical model f(x), where x - is donor percentage in chimera, f(x) - difference between real recipient percentage (100-x) and calculated for locus with loss of allele amplification ((100-x)/2:((100-x)/2+x))*100% (Fig.1c) to test this hypothesis. The highest difference in chimerism calculation (about 17%) is expected in about 40% of donor and 60% recipient percentage. For Patient 2 the STR analysis detected only one LOH locus in DNA samples from cerebrospinal fluid during CNS relapse before and after BMT. However this patient has eight homozygous STR loci and complete donor chimerism in bone marrow. To find possible “loss of allele amplification” we used the artificial chimera, the mix of Patient 2 and unrelated random healthy donor DNA (Fig.1d). We used control DNA of Patient 2 (bone marrow before BMT) to check chimerism level deviations in loci of interest. The decreased level of mixed chimerism was detected in two homozygous loci from cerebrospinal fluid DNA-sample. The decreased estimate for chimerism correlated with proposed math model. For the same STR loci in control DNA no chimerism deviations were detected in mixed artificial chimera (Fig.1e).Conclusions: The LOH loci pattern might be preserved during long period of disease for certain malignant clone. Mixed chimerism or artificial mixed chimera can be useful in finding of homozygous loci with loss of one allele amplification. We suppose homozygous loci are the preferable subject for investigating LOH mechanisms. Loci with “loss of allele amplification” might be the used as malignant cells population marker.This study was supported by the Russian Foundation for Basic Research (RFBR) grant 18-015-00399a. DisclosuresRisinskaya:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Chabaeva:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Kulikov:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding.

Clinical diagnosis of adult patients with acute megakaryocytic leukemia.

Acute megakaryocytic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), which is challenging to diagnose due to frequent myelofibrosis (MF) and a low percentage of blast cells. In the present study, clinical characteristics and experimental observations in 9 adult patients diagnosed with AMKL, who were recruited by the Sino-U.S. Shanghai Leukemia Co-operative Group, were analyzed in order to summarize the diagnostic experience and provide recommendations on diagnosing AMKL. All the patients were diagnosed according to the 2008 World Health Organization diagnostic criteria. The mean age of the patients with AMKL was 59 years (range, 53–68 years). A total of 8 patients had different degrees of anemia, and 2 patients had <5% marrow blasts present in the bone marrow; however, the percentage of positive cells with cluster of differentiation (CD)41 and CD61 expression was >20%, as demonstrated by flow cytometry. A total of 6 patients were positive for platelet-specific antigens, as indicated by immunocytochemistry. Furthermore, 7 patients presented with moderate or marked MF, as demonstrated by a bone marrow biopsy. Karyotypic analysis indicated that 6 patients had abnormal karyotypes. Only 1 patient exhibited the Janus kinase 2V617F mutation. Treatment efficiency was notably poor, with a median survival time of 6.0 months (range, 1.1–24.0 months). In conclusion, the diagnosis of AMKL requires a combination of the results of bone marrow smears and bone marrow biopsy, immunophenotype or immunohistochemistry. We recommend that routine immunophenotypic analysis should include the CD41 and CD61 markers for diagnosing acute leukemia when bone marrow morphology does not indicate the diagnosis.