Immunophenotype Of Blast Cells Research Articles

The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia.

Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment.

Prognostic Role of CD200 in Acute Lymphoblastic Leukemia Patients.

Overexpression of CD200 in ALL patients indicates that it may be useful in the characterization of leukemia initiating cells (LIC). We aim at investigating the expression pattern of CD200 on leukemic B cells and the correlation of CD200 expression with various clinical and laboratory findings in 62 newly diagnosed acute lymphoblastic leukemia patients. All patients were subjected to full history taking, a thorough clinical examination, and laboratory investigations, which included complete blood count (CBC), BM aspiration, immunophenotyping of blast cells, and CD200 expression. There is a higher statistically significant mean value of CD200 expression among the cases (66.15 ± 23.08) than the control group (0.37 ± 0.2) (p value ≤ 0.001). CD200 expression shows a significant correlation with total leucocytic count and hemoglobin level (p = 0.001, 0.03, respectively). This study showed that CD200 expression was expressed in 100% of the patients. Correlations between CD200 expression and different laboratory data of patients revealed that there was an impact of CD200 on different diagnostic findings. After the follow-up of the patients, we found that the use of PRISM function of the software could add value to the detection of minimal residual disease.

Diagnosis of acute myelomonocytic leukemia in a patient with extramedullary testicular lesion

A complex clinical case of acute myelomonocytic leukemia with extramedullary lesion of the testis is presented. patient yu., born in 1968, applied to the National Medical Research Centre for Oncology (Rostov-on-don) after an injury to the inguinal region. ultrasound was performed: in the right testicle in the middle third, a mass of 30 × 23 × 16 mm was revealed. A biopsy was performed: the morphological picture is characteristic of a typical seminoma. Orchofuniculectomy was performed on the right. Histopathological conclusion: the morphological picture is more characteristic of a typical seminoma, but does not allow excluding lymphoma. In order to differentiate between a germ cell tumor and a lymphoproliferative disease, an immunohistochemical study of the tumor tissue, a morphological and immunophenotypic study of the bone marrow were performed. According to the immunohistochemical data, the morphological picture and immunophenotype of tumor cells are characteristic of extranodal NK/T-cell lymphoma of the testis with Cd4 co-expression. However, according to the myelogram data, 20 % of morphologically heterogeneous blast cells were found: with round or bean-shaped nuclei, delicate mesh structure of chromatin, 1–2 nucleoli and a monocytoid form of nuclei with indistinct nucleoli. The cytoplasm of varying basophilia degrees, vacuolized, with delicate azurophilic granularity. The content of the monocytoid population was increased (19 %), represented mainly by promonocytes, which corresponds to acute myelomonocytic leukemia. According to flow cytometry, the immunophenotype of blast cells corresponds to acute myeloid leukemia with Cd56 co-expression. In connection with the new data obtained, the histological preparation was revised again with the expansion of the immunohistochemical study. Result: morphological picture and immunophenotype of tumor cells are characteristic of acute myelomonocytic leukemia with extramedullary lesions of the right testicular tissue. final diagnosis: acute myelomonocytic leukemia with extramedullary lesion of the right testicle, with Cd56 co-expression. The presented clinical case showed the need to use a wide range of diagnostic techniques to determine the nature of the disease. The results of morphological and cytometric studies of the bone marrow were decisive in establishing the diagnosis of M4 acute myeloid leukemia with extramedullary lesions of the right testicle in this patient.

Machine Learning Based Analysis of Relations between Antigen Expression and Genetic Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukaemia.

Flow cytometry technique (FC) is a standard diagnostic tool for diagnostics of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) assessing the immunophenotype of blast cells. BCP-ALL is often associated with underlying genetic aberrations, that have evidenced prognostic significance and can impact the disease outcome. Since the determination of patient prognosis is already important at the initial phase of BCP-ALL diagnostics, we aimed to reveal specific genetic aberrations by finding specific multiple antigen expression patterns with FC immunophenotyping. The FC immunophenotype data were analysed using machine learning methods (gradient boosting, decision trees, classification rules). The obtained results were verified with the use of repeated cross-validation. The t(12;21)/ETV6-RUNX1 aberration occurs more often when blasts present high expression of CD10, CD38, low CD34, CD45 and specific low expression of CD81. The t(v;11q23)/KMT2A is associated with positive NG2 expression and low CD10, CD34, TdT and CD24. Hyperdiploidy is associated with CD123, CD66c and CD34 expression on blast cells. In turn, high expression of CD81, low expression of CD45, CD22 and lack of CD123 and NG2 indicates that none of the studied aberrations is present. Detecting aberrations in pediatric BCP-ALL, based on the expression of multiple markers, can be done with decent efficiency.

IMMUNOPHENOTYPIC AND CYTOGENETIC FEATURES OF ACUTE LEUKEMIA IN CHILDREN OF THE ARKHANGELSK REGION: A RETROSPECTIVE STUDYN: A RETROSpECTIVE STUDY

Background. For the proper diagnosis and optimal prognosis of acute leukemia (AL), a complex of clinical and laboratory methods is used. Among them, chromosomal analysis plays an important role. Cytogenetic markers of leukemic cells are widely used in clinical settings. WHO classification of Tumors of Haematopoietic and Lymphoid Tissues is based on a combined analysis of clinical, cytomorphological, immunophenotypic and cytogenetic findings. Immunophenotyping of blast cells using monoclonal antibodies has significantly improved the diagnosis of leukemia by detecting the grade of tumor differentiation. Aim: based on the results of immunophenotypic and cytogenetic examinations, to describe the structure of AL and to study the effect of cytogenetic abnormalities on the prognosis of acute leukemia in the child population of the Arkhangelsk region. Material and Methods. Archived data were used to analyze the results of cytogenetic and immunophenotypic examinations of all children aged 0–17 years in the Arkhangelsk region, who were diagnosed with AL in the period from January, 2004 to december, 2018. Results. Immunophenotypic examination of bone marrow blast cells in patients with AL revealed that B-linear lymphoblastic leukemia was the most common (74.8 %). Among the quantitative anomalies, hyperploidy was the most common (additional chromosomes 4, 10 and 17 were observed). The t(12; 21) was the most frequent structural chromosomal abnormality. The philadelphia chromosome (ph) (9;22) was found in 6.5 % of cases. The highest risk of death was observed in AL patients with the acute myeloid leukemia phenotype (p<0.001). The overall survival in AL patients significantly improved over the study period (p<0.001). Conclusion . No specific differences in the structure of chromosomal abnormalities were detected. All chromosomal abnormalities which usually have negative effects on the prognosis of the disease also showed negative impacts on the prognosis. Survival of AL patients significantly increased over the study period. The lowest survival was in patients with the acute myeloid leukemia phenotype.

Імунофенотиповий профіль бластних клітин як маркер діагностики рецидивів гострої лімфобластної лейкемії у дітей

Immunophenotyping of leukemia cells was studied in this work; minimal residual disease was monitored among children with acute lymphoblastic leukemia under conditions of relapse and complete remission after the application of ALLIC-BFM 2009 cytostatic therapy. The study showed that after application of ALLIC-BFM 2009 therapy, 88% of children had complete remission, and 12% had relapses. Among patients with relapses, the number of blast cells in the bone marrow was at a high level (more than 6%). Monitoring of patients during therapy established an increase in the minimal residual disease level of more than 1% after treatment in patients with recurrent disease. Immunophenotyping of blast cells among patients with relapse showed the expression of linear independent antigens HLA (93%), Auti-TdT (91%), CD10 (78%), CD38 (91%) and CD34 (57%) and B-linear antigens: CD19, CD22, CD58, CD79a, the highest expression was found for the CD19 antigen. A low level of expression of CD45 (28%) was recorded with relapses of acute lymphoblastic leukemia and high (89%) level was with complete remission of the disease. We did not detect expression of antigens characteristic of T-cell acute lymphoblastic leukemia in bone marrow of patients with acute lymphoblastic leukemia, both with relapses and with remission. At the same time, the expression of myeloid antigens (CD33 and CD13) was noted among acute lymphoblastic leukemia patients. Among patients, the incidence of acute lymphoblastic leukemia was the most pronounced in children aged from 3 to 6 years – 37 patients (35.2%) and aged from 6 to 9 years – 26 (24.8%) patients. The highest accidence was found among patients with chromosomal translocation TEL / AML – 22 (21%) patients with a median age 5 years. In second place, the frequency of mutations is the translocation of E2A / PBX1. BCR / ABL translocation was less common. It was noted in 1.9% of patients, but the expression of this gene indicated a bad course of the disease, as patients after cytostatic therapy under the ALLIC BFM 2009 program had a recurrence. Recurrence was also observed in patients with TEL/AML chromosomal translocation. Determination of minimal residual disease showed its increased level in patients with chromosomal aberrations BCR / ABL and TEL/AML throughout the treatment phase. In addition, patients in these groups were diagnosed with initial leukocytosis followed by leukopenia after a course of chemotherapy. Patients of all groups showed a decrease in hemoglobin. The biggest changes in clinical and laboratory parameters were found between patients with chromosomal translocations BCR/ABL and TEL/AML, as evidenced by the development of relapses in patients of these groups. The low level of association between karyotype disorders, with the formation of AF4/MLL and E2A/PBX1, and clinical and laboratory parameters in patients with acute lymphoblastic leukemia may indicate that the isolated clonal disorders are independent prognostic factors for the course of the disease

RELATIONSHIP OF BLAST CELL IMMUNOPHENOTYPE WITH REMISSION IN ACUTE MYELOID LEUKEMIA IN CHILDREN

Introduction. Acute myeloid leukemia (AML) is a clonal disease of the blood system that occurs as a result of mutations in the genome of hematopoietic progenitor cells. As a result of mutations, the linear differentiation of hematopoietic cells is replaced by the proliferation of malignant myeloid progenitors. Currently, the risk group for AML in children is determined mainly by the presence of specific gene and chromosomal abnormalities and an increased level of peripheral blood leukocytes. The features of the immunophenotype of blast cells can also influence the course of the disease.The aim of the work is to assess the relationship between the immunophenotypic parameters of blast cells and the probability of achieving remission in children with AML.Materials and methods. The study included 109 patients aged 3 months to 17 years who received treatment according to the AML BFM 87, AML BFM 2004, NII DOG AML 2007 и NII DOG AML 2012 protocol in the period from 1991 to 2020.Results. The study showed the relationship between the probability of achieving remission and the presence of markers CD33, CD19 and CD14 on tumor cells. Expression of lymphoid antigen CD19 on blasts was associated with a higher rate of remission (73.0 % vs 95.5 %, p = 0.027). The absence of the linearly associated myeloid marker CD33 negatively correlated with the remission rate (61.1 % vs 87.7 %, p = 0.007). In the presence of monocyte antigen CD14 on blasts, the probability of achieving remission was low (95 % vs 50 %, p = 0.013).Conclusion. Characteristics of the immunophenotype of tumor cells in AML in children are associated with the probability of achieving remission.

Treatment of acute lymphoblastic leukemia in adolescents and young adults: the experience of the Moscow–Berlin

One of the main risk-factors of acute lymphoblastic leukemia (ALL) is age. The most favorable age group is children from 2 to 9 years old, and the adolescents and young adults have the worst prognosis. Treatment of ALL at adolescents and young adults is a unique problem of modern hematology now. This work presents the analysis of 368 cases of patients with primary ALL 15–18 years old, which were registered in the database of Russian-Belarussian group from April 2002 to November 2014. The aim of the analysis was the estimation of effectiveness of different generations of protocol for adolescents and prognostic significance of different factors to reveal the ways of future therapy optimizing in this age group. Event-free survival of adolescents 15–18 years old according our study was 56 ± 5 % in ALL-MB-2002 study and 57 ± 6 % in ALL-MB-2008 study. We did not received any differences in survival based on immunophenotype of blast cells. But the risk group analysis showed the worst results at patients with ALL from pre-B-precursor cells of high risk. No difference was revealed according the gender. One of the general problems remains high toxicity of therapy at patients of this age group. Treatment related mortality (TRM) was 13.2 and 12 % at ALL-MB-2002 and ALL-MB-2008 accordingly. Main cause of deaths remains infection.

Aggressive Natural Killer Cell Leukemia Secondary to Hodgkin Lymphoma: a Case Report and Review of the Literature

A 32-year old Chinese woman presented with high fever (39C), lymphadenopathy, hepatosplenomegaly, shortness of breath and general fatigue after initial remission of Hodgkin lymphoma (HL). The immuno-phenotype of blast cells found in her peripheral blood and pleural fluid suggested abnormal Natural Killer (NK) cells (CD2+CD3- CD56+CD45RO+HLADR+CD94+ with high Ki67). Her serum EB virus DNA was over 1.0×107IU/mL. Combining her clinical symptom and laboratory data, the patient was diagnosed with Hodgkin Lymphoma, secondary NK cell leukemia and haemophagocytic lymphohistocytosis (HLH). To date, this is the first reported case that developed secondary ANKL after the treatment of Hodgkin Lymphoma. Secondary treatment-related cancers are a major problem in HL survivors. Thus, novel treatment strategies for HL should aim at a reduction of chemotherapy and radiation therapy in order to reduce the risk for development of therapy-related malignancies. In conclusion, improvements of combined therapy as well as close monitoring during clinical follow up could warrant the improved overall survival of HL and prevent fatal complications.

Immunophenotypic Characteristics of CD34+ Cells According to Genetic Markers in Acute Myeloid Leukemias with Normal Karyotype.

AbstractAbstract 2554 Introduction:The Acute Myeloid Leukemias of Normal Karyotype (AML-NK) are a subgroup of hematologic malignancies with high heterogeneity, having some of them genetic markers with prognostic value. There is little information about the potential influence of different genetic markers in AML-NK on the immunophenotype of blast cells, particularly of immature populations. Objective:The aim of the present study is to analyze the potential influence of the presence or absence of different genetic markers (FLT3-ITD, mutNPM1) and gene expression levels of BAALC, ERG, EVI1, MN1, PRAME, FOXO3A and WT1) on the immunophenotype of AML-NK, focusing on the phenotypic characteristics of immature blast cells (CD34+ cell compartment). Material and methods:We included in the study 57 cases of de novo AML-NK confirmed by conventional cytogenetics and absence of known molecular alterations by FISH (chromosomes 5,7,8, and 11q23 probes), in which extensive molecular studies as well as a large panel of four colour multiparametric flow cytometry immunophenotype (CD34, CD45, CD19, cyCD79a, nTdT, cyMPO, cyCD3, CD7, CD2, CD56, CD65, 7.1, CD11b, CD13, HLADR, CD117, CD61, CD33, CD123, CD36, CD64, CD14, CD71, Glycophorin A, CD15, CD16) were performed at diagnosis. The CD34 and CD45 antigens were included in all four colour combinations.In 15/57 patients the FLT3 gene and 28/57 cases the NPM1 were mutated. The fold change in expression of the target genes relative to an internal control gene (ABL) was expressed as: Fold Change (RQ): = 2(–ΔCt sample) / 2(–ΔCt calibrator), where the DCt (Ct target gene – Ct ABL) of each patient sample is compared with a calibrator sample (mean expression of five bone marrow from healthy donors), using the DDCt method.The median gene expression was 1.228, 0.392, 3.253 and 0.758 for BAALC, FOXO3A, MN1 and ERG and the 75th percentile was 0.361, 33.87 and 259.77 for EVI1, PRAME and WT1 respectively.The threshold to define positivity for an antigen was set at 20% of blasts population analyzed. In addition, the complete immunophenotype was analyzed on the CD34+ blast cells when we identified a CD34+ blast cell subpopulation greater than 3% of the total blast cells, in order to include the minor subpopulations. The software SPSS v19 was used for the statistical analysis. Results:Upon comparing FLT3-ITD+ vs FLT3-ITD- patients, no differences were observed in the overall incidence of CD34+ and/or CD117+ cases. Nevertheless, if we considered cases with small CD34+ subsets (3%–20% of CD34+ blast cells) the frequency was higher among FLT3-ITD+ patients (40% vs 4%). Upon analyzing the phenotypic profile of the CD34+ cells according to FLT3-ITD status, in patients positive for this marker the CD34+ cells more frequently expressed CD7 (p=0,02) and CD36 (p=0,04).The same comparison was done according to NPM1 status (mutated vs non mutated). The incidence of CD34+ cases was significantly lower while the number of CD117+/CD34- blasts was significantly higher in the mutated subgroup. No differences were observed in the phenotypic profile of the CD34+ cells.For BAALC and MN1 expression, patients were divided according to the median value into two categories: high and low expression. In both markers, the high expression was associated with increased frequency of CD34+ cases (75 vs 17%, p=0,00) and (71 vs 21%, p= 0,01), respectively. The phenotypic profile of CD34+ cells did not differed between high and low groups.Patients with high ERG expression showed a tendency to be associated with increased frequency of CD34+ cells, but differences did not reached statistical significance (p=0,08). Of note, CD34+ cells expressed cMPO less frequently among the ERG high patients.We have no detected differences in the expression of blasts according to EVI1 or FOXO3A expression. Interestingly, in cases with low level of expression of these two markers, the CD34+ blast cell compartment more frequently expressed CD7 (p= 0,01 and p= 0,02, respectively).Finally, PRAME and WT1 expression were not related with the CD34 expression neither with the phenotypic profile of the CD34+ blasts. However, high WT1 was associated with a significantly higher number of CD117+/CD34- cells (p<0.01). Conclusion:The presence of FLT3-ITD and NPM1 mutations as well as the level of expression of relevant genes in AML-NK patients correlates with the CD34 expression on blast cells as well as the phenotypic profile of this immature cell subset. Disclosures:No relevant conflicts of interest to declare.

Concomitant aberrant methylation of p15 and MGMT genes in acute myeloid leukemia: association with a particular immunophenotype of blast cells

In this study, methylation-specific polymerase chain reaction (MS-PCR) was used to define the methylation status of the target promoter sequences of p15 and MGMT genes in the group of 21 adult patients with acute myeloid leukemia (AML). The incidence of aberrant hypermethylation of p15 gene (71 %) was higher comparing to MGMT gene (33 %), whereas concomitant methylation of both genes had 24 % of the patients. Although the incidence of cytogenetic abnormalities between the groups with a different methylation status of p15 and/or MGMT genes was not significantly different, we observed general trend of clustering of abnormalities with adverse prognosis into groups with concomitant hypermethylation of both genes and only p15 gene. Also, we showed that AML patients with concomitant methylation of p15/MGMT genes had a higher proportion of leukemic blast cells characterized with specific expression of individual leukocyte surface antigens (CD117(+)/CD7(+)/CD34(+)/CD15(-)), indicating leukemic cells as early myeloid progenitors. Although we could not prove that hypermethylation of p15 and/or MGMT genes is predictive parameter for response to therapy and overall survival, we noticed that AML patients with comethylated p15/MGMT genes or methylated p15 gene exhibited a higher frequency of early death, lower frequency of complete remissions as well as a trend for shorter overall survival. Assessing of the methylation status of p15 and MGMT genes may allow stratification of patients with AML into distinct groups with potentially different prognosis.

Diagnostic work-up for clinical and prognostic assessment of acute leukaemia

Acute leukaemia is a neoplastic disorder of haematopoietic precursors characterized by a rapid clinical course and a relatively high early death rate in spite of recent therapeutic progress. Prompt and reliable diagnostic and prognostic assessment has a favourable impact on patient outcome. Diagnostic suspicion relies on signs and symptoms of bone marrow failure or quali-/quantitative abnormalities of blood cells, which are accurately detected by modern automated counters. Cytomorphological examination of a stained blood and bone marrow film plays a key role in the diagnostic process, with relevant additional information coming from immunophenotyping of blast cells. Cytogenetic and molecular genetic data are the basis of prognostic stratification. In this context, a coordinated intervention of specialized laboratories combining solid expertise in each field involved in the diagnostic work-up is essential; at the same time, the availability of all the required technologies at the same hospital structure would probably lack efficiency due to the low number of tests performed. The organization of laboratory networks, either at regional or national level, especially in the context of clinical trials, may offer a great opportunity for centralization of more sophisticated technologies in reference laboratories, each highly specialized in a particular field of investigation and all interconnected. The challenge for laboratory haematology is the reorganization of clinical and scientific activity according to this model, without loosing educational potential in favour of new generations of medical doctors, haematologists, biologists and laboratory technicians.

WT1 Protein Expression in Myeloid Malignancies: An Immunohistochemical Study

Abstract 4886The Wilms tumor 1 (WT1) gene encodes for a zinc-finger protein that may act both as a tumor suppressor and as an oncogene. It is widely expressed in normal tissues, especially in the urogenital system during fetal development. WT1 is a transcriptional regulator of genes involved in the cell growth and metabolism, such as c-Myc, bcl-2 and epidermal growth factor receptor (EGFR). Its abnormal mRNA overexpression in myeloid malignancies has been exploited as minimal residual disease marker. WT1 mutations have also been described in a percentage of acute myeloid leukemia (AML) with a normal karyotype. It has been reported that these patients had a bad outcome. Few data are available on WT1 protein expression in myeloid neoplasms.WT1 expression was investigated in bone marrow samples obtained from 61 patients (54 AML, 1 MPN, 5 MDS and one case with myeloid sarcoma) and 5 controls. Blast cell immunophenotype and cytogenetics were available for each case. WT1 mutations were investigated in 14 AML cases. Immunohistochemistry procedures were performed on formalin-fixed paraffin embedded whole tissue sections with primary monoclonal antibody against WT1 (WT1 MxH 6FH2 clone, DAKO, Glostrup, DK). The staining intensity was graded on a 0 to 3 scale (0, negative; 1, weak; 2, moderate; and 3, intense staining) and the percentage of stained cells was scored. To obtain the final score, both values (staining intensity and percentage of positive cells) were multiplied. Cases with WT1 score under 10 were considered as negative. We also analyzed WT1 mRNA gene expression on the same cases by real-time PCR as previously described (Cilloni D et al, J Clin Oncol 2009).Relationship between quantitative variables was evaluated by non-parametric correlation analysis (Spearman correlation test). Two-sided p value under 0.05 was considered significative.In normal bone marrow, WT1 immunohistochemical expression was restricted to the cytoplasm of occasional megakaryocytes and endothelial cells, whereas no nuclear staining was observed neither in hematopoietic cells nor in endothelial and circulating lymphoid cells. Immunohistochemical WT1 expression was found in 43 out of 61 (70.5%) cases. The mean value of mRNA WT1 expression was 3812.3 copies (standard deviation (SD):7163.9). We observed a positive correlation between WT1 score and mRNA WT1 expression (p<0.0001; Spearman’s rho coefficient=0.611). Moreover, this correlation was maintained when we separated normal and complex karyotype groups (Normal: p<0.0001; Spearman’s rho coefficient=0.755; Complex: p=0.023; Spearman’s rho coefficient=0.318). Nevertheless, in four cases (3 AML and one CMML) with no WT1 protein expression, the WT1 mRNA levels were above 1800 copies (Normal<200). In the three cases with WT1 mutations the results were also discordant.In myeloid malignancies, WT1 protein expression showed some degree of correlation with the WT1 mRNA levels. It remains to be investigated the mechanisms which can explain the discordances and if these findings could provide prognostically relevant information. Disclosures:No relevant conflicts of interest to declare.

WT1 Gene Expression Limited Application for MRD Monitoring in AML Patients Carrying MLL Rearrangements.

Abstract 4700 StatementWT1 gene expression (GE) is widely used for monitoring of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients. Predictive value of WT1 GE in MLL-positive AML is not clear. Comparison of WT1 GE and fusion genes (FG), detected at RNA and DNA levels is rarely done. Here we present comparison of MRD data, assessed by WT1 GE, FG transcripts and MLL fusions alleles (MFA). Materials and methods27 samples from 5 MLL-positive AML patients (pts) were analyzed. Median age was 13 years (range 0.8-58 years). 5 samples were obtained at diagnosis, 21 samples during follow-up treatment, 1 at diagnosis of relapse. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics. Presence of MLL rearrangements was detected by FISH, reverse-transcriptase PCR (RT-PCR) and confirmed by long-distance inverse PCR. Among 5 pts there were two MLL-MLLT4-positive cases, two MLL-MLLT10-positive. One patient carried MLL-MLLT3 fusion gene. Quantification of MFA in genomic DNA by patient-specific primers-probe combinations and detection of FG transcript kinetics in RNA/cDNA during treatment were performed by RQ-PCR as previously described (T. Burmeister et al, Leukemia 2006, 20; J. Gabert et al, Leukemia, 2003, 17, respectively). Due to high concordance rate (96%) between qualitative results obtained by FG transcript and MFA detection, these results were combined into one group named as standard methods (SM) of MRD monitoring. Sample was defined as positive if only one result obtained by SM was positive. Sample was defined as negative if both results assessed by FG transcript and MFA were negative. Quantification of WT1 was performed in bone marrow (BM) samples by real-time quantitative PCR (RQ-PCR) with normalization to ABL gene. Additionally, BM samples taken from 14 pts without hematological malignancies were investigated. WT1 threshold level (TL) calculation was based on ROC curve analysis. TL was defined as value with maximal sensitivity and specificity. ResultsROC analysis of WT1 GE revealed an area under curve (AUC) of 0.890 (95% CI 0.800-0.990). TL was set at 32 with sensitivity of 62% and specificity of 100%. Value of TL was equal to upper level of WT1 normalized copy number (NCN) in BM of 14 pts without hematological malignancies (median 10, range 0-32 WT1 copies/104ABL copies). In AML pts it was detected 13 WT1-positive samples (48%). 21 samples (78%) were positive by SM. 6 samples (22%) were negative both by SM and WT1 GE. Discrepant results were found in 8 samples (30%) (table 1). These discrepant results were observed in follow-up samples only. Qualitative concordance between SM and WT1 GE was 70% in our series.Table 1WT1 positiveWT1 negativeSM Positive138SM Negative06 ConclusionsBased on the ROC curves analysis it was not found particular TL of WT1 NCN that clearly distinguishes MRD-negative pts from those with positive MRD, detected by SM. Applicability of WT1 NCN quantification for MRD monitoring in the assessed MLL-rearranged AML samples appears to be limited because of relatively high percentage of false-negative samples. Discrepant results could be explained by high variability of WT1 GE during antileukemic therapy, and presence of WT1 GE in normal BM. Thus WT1-negative results should be considered as truly MRD-negative with great caution and have to be confirmed by FG transcript and/or MFA quantifications. Disclosures:No relevant conflicts of interest to declare.

Plasmablastic crisis of Philadelphia chromosome-positive chronic myeloid leukemia

Dear Editor, The cytogenetic hallmark of chronic myeloid leukemia (CML) is the Philadelphia chromosome (Ph), the consequence of the reciprocal translocation t(9;22)(q34;q11.2) that results in the BCR-ABL1 fusion gene, located in the Ph chromosome [1]. The natural history of untreated CML is an initial indolent chronic phase which is followed by a blast phase either suddenly or through an accelerated phase. Blast crisis may be diagnosed when blast cells are ≥20% of the peripheral blood WBC or the nucleated cells of the bone marrow or when there is an extramedullary blast proliferation [1, 2]. In approximately 70% of cases the blast lineage is myeloid while in 20–30% of cases the blasts are lymphoblasts [1, 2]. Progression is usually associated with clonal evolution, and at the time of transformation to blast crisis, 80% of patients demonstrate cytogenetic changes in addition to the Ph chromosome such as an extra Ph chromosome, +8, i(17q), +19, t(3;21)(q26;q22), and very rarely t(7;11)(p15;p15) [3]. Plasma cell leukemia (PCL) is an aggressive and rare hematological malignancy which is characterized by circulating plasma cells >2×10/l in peripheral blood or by a plasmatocytosis >20% of blood leukocytes and confers a poor prognosis. It originates as a primary disease or as a secondary leukemic transformation of multiple myeloma [4]. The most frequent features at diagnosis are asthenia, severe anemia, thrombocytopenia, splenomegaly, or hepatomegaly while CD38 and CD138 antigens are excellent plasma cell markers [5, 6]. In the majority of the cases, karyotypes are complex and hypodiploid or pseudodiploid. Common chromosome abnormalities are 14q32 translocations such as t(11;14)(q13;q32), t(4;14)(p16.3;q32), t(14;16)(q32;q23), deletions of the chromosomal regions 17p13, 13q, and 1p21, monosomies of chromosomes 13 and X (in women), trisomy 18, amplification of 1q21, and MYC rearrangements [4–7]. Here, we report for the first time a rapidly fatal case of a PCL crisis in a young patient with Ph chromosome-positive CML. The patient, a 43-year-old male with a 2-month history of persistent fatigue, weakness, and loss of weight was admitted to our department. On physical examination spleen was remarkably enlarged and routine complete blood count revealed leukocytosis with a WBC of 22× 10/l, anemia with a Hb of 7 gr/dl, and thrombocytopenia with platelet count of 30×10/l. Neither hypercalcemia nor proteinuria was observed. Blood smear showed 90% blasts with the appearance of plasma blasts (Fig. 1). Immunophenotyping of blast cells by flow cytometry showed CD45+ (79%), CD38+ (77%), CD34+ (56%), surface κ(−), surface λ(-), cytoplasmic κ+ (90%), cytoplasmic λ(−), and CD20 (−). The cytogenetic analysis of G-banded chromosomes in peripheral blood cells revealed two evolutionary clones, which were positive for Ph chromosome. The karyotype was described as: 46,XY,t(9;22)(q34;q11.2)[4]/32,XY,-2, -3,-4,-5,t(9;22)(q34;q11.2),-10,-11,-12,-13,-15,-16,-17,-18, -20,-22[16]. Unfortunately, the patient developed acute renal failure on the second day after admission and died 2 days later. K. N. Manola (*) Laboratory of Cytogenetics, National Center for Scientific Research (NCSR) “Demokritos”, 15310 Aghia Paraskevi, Athens, Greece e-mail: pmanola@ipta.demokritos.gr

Burkitt lymphoma presenting as posterior reversible encephalopathy syndrome secondary to hypercalcaemia

A 50-year-old Caucasian female presented with fever and increasingly obtunded. Physical examination showed normal blood pressure and no focal neurological signs. There was no organomegaly or lymphadenopathy. A full blood count showed: haemoglobin 86 g/l, white cells 9AE9 · 10/l (with 6% blasts) and platelets 114 · 10/l. A coagulation screen showed evidence of disseminated intravascular coagulopathy. Biochemistry tests revealed hypercalcaemia with a calcium of 3AE91 mmol/l and increased ionized calcium to 2AE05 mmol/l, in association with raised lactate dehydrogenase to 345 iu/l but normal alkaline phosphatase level. The serum free kappa light chain level was increased to 33AE6 mg/l (reference interval: 3AE3–19AE4 mg/l) and there was generalized immunoparesis. Bone marrow aspiration showed hypercellular particles packed with 76% blasts (left upper panel). The blast cells were mostly medium sized showing round nuclei, dispersed chromatin, multiple small nucleoli and scanty basophilic cytoplasm that was sometimes vacuolated. Trephine biopsy showed hyperplastic marrow that was diffusely replaced by a monotonous population of medium-sized blasts with frequent mitotic figures. Blast cell immunophenotyping by flow cytometry supplemented by immunohistochemistry demonstrated clonal B-cells (CD19 CD20 CD22 CD79a surface IgM) that expressed CD10 and BCL2, but were negative for terminal deoxynucleotidyltransferase and BCL6. The Ki-67 labelling index was virtually 100%. Interphase FISH on bone marrow blasts showed MYC gene rearrangement (left lower panel) as detected by a dual colour break-apart probe (Abbott Molecular, IL, USA) but negative for IGH/MYC fusion, consistent with MYC translocation other than classical t(8;14). The IGH/BCL2 fusion was negative. The diagnosis was Burkitt lymphoma. Magnetic resonance imaging (MRI) of the brain showed symmetrical posterior cortical and subcortical lesions in axial (right upper panel) and coronal (right lower panel) sections. The cerebrospinal fluid was unremarkable. The patient was started on haemodialysis for acute renal failure (creatinine 463 lmol/l) and the calcium level was normalized after institution of cytotoxic therapy. A repeat MRI scan after treatment showed complete resolution of the brain lesion. The neurological diagnosis was posterior reversible encephalopathy syndrome (PRES) secondary to severe hypercalcaemia. The patient returned to her native country for further management. This case illustrated the uncommon presenting feature of PRES due to hypercalcaemia in Burkitt lymphoma.

MRD Monitoring in AML Patients with MLL-MLLT4 by Quantification of Fusion Gene Transcripts and Genomic Chromosomal Breakpoint Sequences

Statement. The MLL-MLLT4 (former MLL-AF6) fusion gene (FG) is relatively rare genetic abnormality, predominantly found in AML. It averages 3–5% among other MLL rearrangements. Here we present data of MRD monitoring in 2 patients with AML carrying an MLL-MLLT4 rearrangement by 2 approaches: usingFG transcript at RNA/cDNA level in comparison withFG at genomic DNA level.Materials and methods. Patients (pts) were diagnosed according French-American-Britain (FAB) classification. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics, FISH and reverse transcriptase PCR (RT-PCR). RT-PCR products were directly sequenced afterwards. In both cases identification of genomic chromosomal breakpoint sequences within MLL and MLLT4 genes was done by long-distance inverse PCR (LDI-PCR). MRD quantification in genomic DNA was performed using patient-specific primers and probes by real-time quantitative PCR (RQ-PCR). 500 ng of DNA was used per reaction. Standard curve was received by serial 10-fold dilutions of pts' DNA into the DNA isolated from pooled lymphocytes of ten healthy donors. b-actin was used as DNA quality and quantity control. Detection of FG transcript kinetics during treatment was performed by RQ-PCR according to “Europe Against Cancer” recommendations for normalization by using control gene ABL (Gabert J. et al Leukemia, 2003, 17) and for using 10-fold dilutions of plasmids carrying MLL-MLLT4 fragment as source of standard curve. MRD value for cDNA targets were estimated as previously described (Beillard E. et al Leukemia, 2003, 17). Each sample was run in triplicates. According to the treatment design, time-points for MRD estimation were scheduled before each block of treatment. Totally 5 samples were evaluated in each patient (initial and 4 follow-ups). FLT3-ITD status was estimated at the time of diagnosis. Informed consent was obtained in both cases.Patients' characteristics, treatment and clinical outcomeCase # 1Case #2Age5813SexMaleMaleInitial WBC*106/ml5.594.9ImmunophenotypeCD34+CD117+HLA-DR+ CD11c+CD13+CD33+CD65+CD34+CD117+CD13+ CD33+CD45+MPO+Cytogenetics46, XY, del(5)(q?), der(5)t(5;6;11) (q22;q15q27;q23), der(6)t(5;6) (q22;q15), del der(11)46, XY, t(6;11)(q27;q23)FISH with LSI MLLMLL deletionMLL splitRT-PCRMLL-MLLT4 positiveMLL-MLLT4 positiveMLL-MLLT4 FG transcriptexon 9-exon 2exon 9-exon 2Localization of genomic chromosomal breakpoint within MLL and MLLT4intron 9-intron 1intron 9-intron 1FLT3-ITDNegativeNegativeInduction treatment7+3AIEConsolidation therapy2× HAM 2× HDAC1× HAM 1× FLAG 1× HAEMaintenance−+Duration of therapy, months87Achievement of CR++OS, months87EFS, months65Current statusAlive in CRAlive in CRResults. Despite of achievement of CR, MLL-MLLT4 FG transcripts were detected in every sample tested after induction and consolidation chemotherapy by RQ-PCR. MRD value in case #1 in cDNA was fluctuated significantly within 2 log. Although in case #2 there was successive reduction from 260% at the beginning of treatment till 0.7% before maintenance therapy (after HAE block). Limited dilution series of a MLL-MLLT4-positive RNA into RNA of ten healthy donors showed a sensitivity limitation of 1E–05. For quantification of genomic chromosomal breakpoint sequences b-actin was amplified in each well. Deviation between Ct values of b-actin in different wells did not exceed ±2.0. In case # 1 the standard curve of the RQ-PCR assay for MLL-MLLT4 FG had slope of −3.19. Correlation coefficient was 0.987. Quantitative range of this assay was 1E–04 and sensitivity 1E–5. It was also observed a considerable variation of MRD levels in genomic DNA during treatment, like it was observed in MRD monitoring by FG transcripts. Fluctuations run up to 2.5 log. In case #2 the standard curve of the RQ-PCR assay for MLL-MLLT4 had a slope of −3.63 with correlation coefficient 0.992. Quantitative range of this assay was reached 1E–04 with sensitivity 1E–05. MRD level in this patient constantly decreased.Conclusions. The same tendency has been shown in each patient: fluctuations of MRD levels (2.5 log in case #1) and successive reduction (case #2). Results received at RNA/cDNA level and in genomic DNA cannot substitute each other, but they can be used as additives. It has been demonstrated that quantification of MLL-MLLT4 FG in genomic DNA is precise and suitable for MRD monitoring.

A Novel Translocation (4;12)(q11;q13) in an Acute Promyelocytic Leukemia-Like Case Lacking RARα Rearrangements.

We report a novel chromosomal translocation t(4;12)(q11;q13) in a 37-year-old male with acute promyelocytic leukemia (APL)-like morphologic changes but lacking RARα rearrangements. His blast cells had some morphologic features evocative APL such as heavy azurophilic granules, bundles of Auer rods except regular round or oval nuclei. Immunophenotype of blast cells showed positivity for CD13 and CD33, and negativity for CD34 and HLA-DR which were compatible with the diagnosis of APL. Disseminated intravascular coagulation (DIC) was present. Chromosome study of BM cells showed that 29 out of 35 metaphases had a consistent karyotype of 46, XY, t (4;12)(q11;q13) which was confirmed by chromosome painting with whole chromosome paint probes 4 and 12. However, fluorescence in site hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR) and multiplex RT-PCR did not demonstrate any evidence for RARα rearrangements including PML-RARα, PLZF-RARα, NuMA-RARα, NPM-RARαand STAT5b-RARα fusion genes. Sequential treatment with arsenic trioxide and chemotherapy had no effect, he died of bleeding due to DIC. In view of the fact as mentioned above, this case should be rated as transitional M2–M3.

The Prognostic Impact of Blast Cell Immunophenotyping in Intermediate Risk AML Patients at Diagnosis.

The most important prognostic factor in current clinical management of acute myeloid leukemia (AML) is the leukemic karyotype, identifying patients with relatively favorable, intermediate and adverse prognosis. Unfortunately, the majority of patients are assigned to the intermediate risk group although they represent a broad spectrum regarding prognosis. While molecular mapping may diminish this problem in the future, an approach to prognostic stratification of patients within the intermediate risk group is urgently needed in order to develop risk-adapted treatment strategies and evaluate the efficacy of new treatments. The aim of this retrospective study was to evaluate the prognostic impact of diagnostic multiparametric flow cytometry (MFC) in the cytogenetic intermediate risk group of AML patients. Diagnostic samples were obtained from 162 AML patients (median age 66 years, range 0–88; cytogenetic risk group favorable/intermediate/adverse/unknown 11/91/17/43; bone marrrow/blood 141/21). Four-color flow cytometry and a panel of 35 monoclonal antibodies was used to characterize the immunophenotype of AML blast cells, defined by side scatter and CD45 expression. For each antigen in the panel, mean fluorescence intensity (MFI) of the blast cell population was recorded. For each antigen, the expression pattern was characterized as homogeneous or heterogeneous. Heterogeneous antigen expression was defined as the presence of two or more subpopulations, within the blast cell gate, expressing the antigen in question with distinctly different intensities. Flow cytometry data, biological and clinical variables for the cytogenetic intermediate risk group and for patients with unknown cytogenetic risk were analysed by univariate Cox regression analysis and selected variables were analysed by multivariate Cox regression analysis (backward likelihood-ratio statistics, p<0.05 considered significant) with overall survival as outcome variable. Four conventional variables and four blast cell phenotype variables significantly predicting inferior survival were identified: FAB type other than 0/1 (p<0.03), presence of extramedullary disease (p<0.007), high white blood count (p<0.0005) and high age (p<0.0005); a heterogeneous CD117 expression (p<0.009), heterogeneous CD90 expression (p<0.03) as well as high MFI of CD56 expression (p<0.0005) and low MFI of CD58 expression (p<0.001) for CD45/SSC gated blast cells. The prognostic model identified two subsets of cytogenetic intermediate risk patients with a median overall survival of 0.1 and 1.5 years respectively. The subset of cytogenetic intermediate risk patients with high risk according to the prognostic model had an outcome inferior to that of the cytogenetically defined adverse risk group. This prognostic model was also valid for patients with unknown cytogenetic risk and for patients assigned to the favorable and adverse risk groups by cytogenetics. Informative MFC was applicable in 96% of the cases analysed. The prognostic model should be evaluated in a prospective clinical multicentre trial.

Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression.

Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy.