The Expression and Clinical Significance of PRAME Gene in Acute Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4225-4225
Author(s):  
Rong Fu ◽  
Kai Ding ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Clinical Significance ◽  
Blood Count ◽  
Mononuclear Cells ◽  
Residual Disease ◽  
White Blood Count ◽  
Gene Expressions ◽  
Healthy Donors ◽  
Blast Cells

Abstract Objective To investigate the expression of PRAME (preferentially expressed antigen of melanoma) gene in acute leukemia and its clinical significance in monitoring prognosis, detecting minimal residual disease (MRD) and gene immunotherapy. Methods The expression of PRAME gene mRNA in bone marrow mononuclear cells is measured by reverse transcriptase polymerase chain reaction in 34 patients with acute leukemia and 12 bone marrow samples of health donors. The relationships between PRAME gene expressions and some clinical data, such as gender, age, white blood count, leukemic immunophenotype, the percentage of blast cells, and the karyotype of chromosome, were also estimated. Results PRAME gene was expressed in 38.2% of all the patients, 40.7% of all the AML patients, which was higher than the 28.6% of ALL patients (p >0.05). There was no expression of PRAME gene in healthy donors. In all the sub phenotypes of AML, the expressive rate of PRAME gene in M3 patients is 80%, which is higher than that in M2 (33.3%) and in M5 (28.6%). The expressive rate of PRAME gene was also positively correlated with the expression of CD15, CD33, and the abnormality in the karyotype of chromosome, but not correlated with age, gender, white blood count and percentage of blast cell in bone marrow. Conclusion PRAME gene is highly expressed in acute leukemia, and could be regarded as a useful tool for monitoring MRD. Differential expression in acute leukemia patients vs. healthy donors suggests that the immunogenic antigens PRAME are potential candidates for immunotherapy in acute leukemia.

Plasma Level of Micro-RNA92-a in Acute Myeloidleukemia and Its Correlation with Prognostic Factors and Therapeutic Response

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4826-4826
Author(s):  
Ahmed Galal
Keyword(s):  
Bone Marrow ◽  
Prognostic Factors ◽  
Plasma Level ◽  
Mirna Expression ◽  
Blood Count ◽  
Therapeutic Response ◽  
Statistical Significance ◽  
White Blood Count ◽  
Control Group ◽  
Blast Cells

Abstract Abstract 4826 Background: Acute myelogenous leukemia (AML) is a clonal, malignant disease of hematopoietic tissues that is characterized by accumulation of abnormal blast cells, principally in the marrow, and impaired production of normal blood cells. MicroRNAs (miRNAs) are short non-coding RNAs of ∼ 21 to 23 nucleotides in length that post-transcriptionally regulate mRNA expression. High-throughput methodologies have shown deregulated miRNA expression in an increasing number of human cancers including colon, breast, lung, thyroid, cervical, ovarian and pancreatic cancers, and miRNA expression patterns have been found to distinguish tumors of different developmental origin, better even than traditional mRNA expression profiling. miR-92a is transcribed from miR-17–92 locus that encode the polycistronic precursor containing seven microRNAs:miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b andmiR-92a, and the human microRNA cluster miR-17–92 is amplified and/or overexpressed in cells of several cancer such as malignant lymphoma, lung cancer, thyroid cancer, and hepatocellular carcinoma. Aim: To assess plasma level of micro-RNA 92a in adult acute myeloid leukemia and to correlate it with prognostic factors and therapeutic response. Patients and Methods: This study was carried out on twenty five subjects with AML admitted to hematology unit in Alexandria university hospital as patients group as well as twenty five healthy subjects as control group. Conventional cytogenetics and FLT3/ITD gene mutation using PCR were done to patients group while measurement of plasma level of miR-92a using TaqMan quantitative RT-PCR and miR-638 as standardization was done to both groups. Therapeutic response was assessed in patients group by doing bone marrow aspirate at day 28 of induction chemotherapy. Results: The ratio of plasma miR-92a to miR-638 in patient group had a mean of 0.239±0.286 while in control group it had a mean of 0.969±0.490 that confirmed statistical significance (p=0.001*). Also there was significant negative correlation between RQ of miR-92a and white blood count in patient group (p= 0.001*). Patients who achieved complete response after induction chemotherapy had a mean RQ higher than non-responder (0.274±0.322 versus 0.073± 0.077 respectively) but with no statistical significance (p=0.212). Also there was no significant correlation between RQ of miR-92a and FLT3/ITD, cytogenetics and bone marrow blasts on admission. Summary/Conclusions: Plasma level of miR-92a is decreased in newly diagnosed AML patients than normal subjects, and it was related to high white blood count. If we correlate these findings with other studies that suggest that microRNAs are packaged inside exosomes that are secreted from cells to be delivered to other cells and function in a new location. Thus, it might be possible that blast cells specifically take in the exosomes that contain miR-92a and, as a result, miR-92a decreases from the plasma. This may justify the possibility of its usage in the prognosis of AML. Our data suggest the potential importance of the microRNAs as noninvasive cancer biomarkers helping in diagnosis, clinical prediction and therapeutic response. Disclosures: No relevant conflicts of interest to declare.

Dynamics of Bcl-2, Bax and ACE Expression In CD34+ Cells of Peripheral Blood and Bone Marrow In Patients with Acute Leukemia During Induction Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4840-4840
Author(s):  
Elena E. Khodunova ◽  
Elena N. Parovichnikova ◽  
Irina V. Galtzeva ◽  
Sergey M. Kulikov ◽  
Valentin G. Isaev ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Acute Leukemia ◽  
Induction Therapy ◽  
Peripheral Blood ◽  
Acute Leukemias ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Blast Cells

Abstract Abstract 4840 The causes of drug resistance in acute leukemias (AL) have been studied very intensively and the key research was done on Bcl-2 family proteins. Last studies have showed that high level Bcl-2 expression in acute leukemia is really associated with drug resistance andpoor prognosis [Haematologica 2007, U. Testa]. It was demonstrated that lower Bax/Bcl-2 ratio (<0,3) was associated with FAB M0-M1 classes (p=.00001), poor-risk cytogenetics and poor prognosis [Blood 2003, G. Poeta]. But there were no studies on the dynamic evaluation of Bcl2 and Bax expression on CD34+ cells during chemotherapy. Renin-angiotensin system and angiotensin concertin enzyme (ACE) influence on leukogenesis is extensively investigated. It was reported that ACE expression on blast cells is high [Leuk Lymphoma 2006, S. Aksu]. Recent publications indicate that primitive hematopoietic precursors have different characteristics regarding ACE: CD34+ACE+cells transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population [Blood 2008, V. Jokubaitis]. But it's still unknown how CD34+ACE+ cells in AL behave on and after chemotherapy. We have studied the dynamics of Bcl-2 and Bax expression by flow cytometry in CD34+ cells of peripheral blood (PB) and bone marrow (BM) in pts with AL. PB and BM samples were collected before treatment, on days +8, +36, only PB - on day + 21. Bcl-2 and Bax were detected on CD34+ cells by flow cytometry using specific monoclonal antibodies: CD34 (8G12, BD), Bcl-2 (100, BD), Bax (2D2, Santa Cruz). ACE (9B9, BD) expression was also evaluated. We calculated 10 000 cells in each sample. 10 pts were included in the study: 4 AML, 6 ALL. The control group comprised 4 healthy donors. At time of diagnosis CD34+ cells number in BM was 38,7%± 9,75, in PB - 38,3%± 8,14 in AL pts, not differing much in AML and ALL, and indicating blast cells population. CD34+ cells numbers in BM and PB of healthy donors were 1,35% and 0,23%, respectively. After induction therapy and WBC recovery (days +36-38) CD34+ cells number in AL pts decreased dramatically in BM to 3,83%±1,51 (p=0,001) and in PB to 0,98%± 0,29 (p=0,0001), indicating the efficacy of chemotherapy. The dynamics of Bcl-2, Bax and ACE expression on CD34+ cells of BM and PB in AL pts are presented in fig.1-6 As seen in the fig.1,2 CD34/Bcl-2 expression in BM is significantly higher (p=0,04) and in PB is similar in AL pts at the diagnosis comparing with donors. It's also worth to note that BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 demonstrating much higher level of antiapoptotic marker in PB cells. On the contrast CD34+ AL cells in BM and PB had similar characteristics regarding CD34/Bcl-2 expression. This expression level decreased substantially in BM at day +36 comparing with day 0 (p=0,04), but it never reached the donors level remaining extremely high and supposing the persistence of antiapoptotic activity in CD34+ cells in AL pts. It did not change at all during chemotherapy in PB cells, being identical to donors characteristics. The fig.2,3 demonstrate that, CD34/Bax expression in BM is almost 3-times higher (p=0,14) and in PB is twice lower (p=0,02) in AL pts in comparison with donors. It's interesting that CD34/Bax expression in leukemic BM and PB cells looks very similar, when in donors we had very low expression in BM and high - in PB. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar in Bax expression. Chemotherapy caused the significant augmentation of CD34/Bax expression in PB on day +8 (p=0,01) and near significant on day +21 (p= 0,09) showing the increased level of “dieing” cells in PB after cytostatic influence. The fig. 5,6 show that CD34/ACE coexpression in BM cells of AL pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AL pts was much lower (p=0,02) than in donors and substantially increased at day +36 almost reaching the donor level. We may conclude that Bcl-2, Bax, ACE expression on CD34+ cells in AL pts and donors significantly differs, the dynamics of expression in AL while chemotherapy shows critical changes in CD34/Bcl-2 expression in BM, CD34/Bax and CD34/ACE in PB. Disclosures: No relevant conflicts of interest to declare.

METHODS OF MULTICLASSICATION OF BONE MARROW CELLS FOR THE DIAGNOSIS OF ACUTE LEUKEMIA AND MINIMAL RESIDUAL DISEASE (ON THE MATERIALS OF THE LABORATORY OF HEMATOPOIESIS OF N. N. BLOKHIN NATIONAL MEDICAL RESEARCH CENTER OF ONCOLOGY)

2020 ◽  
pp. 155-158
Author(s):  
Валентина Викторовна Дмитриева ◽  
Николай Николаевич Тупицын ◽  
Евгений Валерьевич Поляков ◽  
Софья Сергеевна Денисюк
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Medical Research ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Binary Classification ◽  
National Medical ◽  
Cell Class ◽  
Minimal Residual ◽  
Marrow Cells

Применение методов и средств цифровой обработки изображений при распознавании типов клеток крови и костного мозга для повышения качества диагностики острых лейкозов является актуальной научно-технической задачей, отвечающей стратегии развития технологий искусственного интеллекта в медицине. В работе предложен подход к мультиклассификации клеток костного мозга при диагностике острых лейкозов и минимальной остаточной болезни. Для проведения экспериментальных исследований сформирована выборка из 3284 изображений клеток, представленных Лабораторией гемопоэза Национального медицинского исследовательского центра онкологии им. Н.Н. Блохина. Предложенный подход к мультиклассификации клеток костного мозга основан на бинарной модели классификации для каждого из исследуемых классов относительно остальных. В рассматриваемой работе бинарная классификация выполняется методом опорных векторов. Метод мультиклассификации был программно реализован с применением интерпретатора Python 3.6.9. Входными данными программы служат файлы формата *.csv с таблицами морфологических, цветовых, текстурных признаков для каждой из клеток используемой выборки. В выборке представлено девять типов клеток костного мозга. Выходными данными программы мультиклассификации являются значения точности классификации на тестовой выборке, которые отражают совпадение прогнозируемого класса клетки с фактическим (верифицированным) классом клетки. “Эксперимент показал следующие результаты: точность мультиклассификации рассматриваемых типов клеток в среднем составила: 87% на тестовом наборе, 88% на обучающем наборе данных. Проведенное исследование является предварительным. В дальнейшем планируется увеличить число классов клеток, объем выборок различных типов клеток и с уточнением результатов мультиклассификации The use of methods and means of digital image processing in the recognition of types of blood cells and bone marrow to improve the quality of diagnosis of acute leukemia is an urgent scientific and technical task that meets the strategy for the development of artificial intelligence technologies in medicine. The paper proposes an approach to the multiclassification of bone marrow cells in the diagnosis of acute leukemia and minimal residual disease. For experimental studies, a sample of 3284 images of cells was formed, submitted by the Hematopoiesis Laboratory of the National Medical Research Center of Oncology named after V.I. N.N. Blokhin. The proposed approach to the multiclassification of bone marrow cells is based on a binary classification model for each of the studied classes relative to the others. In the work under consideration, binary classification is performed by the support vector machine. The multiclassification method was implemented programmatically using the Python 3.6.9 interpreter. The input data of the program are * .csv files with tables of morphological, color, texture features for each of the cells of the sample used. The sample contains nine types of bone marrow cells. The output data of the multiclassification program are the classification accuracy values on the test sample, which reflect the coincidence of the predicted cell class with the actual (verified) cell class. “The experiment showed the following results: the accuracy of multiclassification of the considered types of cells on average was: 87% on the test set, 88% on the training data set. This study is preliminary. In the future, it is planned to increase the number of classes of cells, the volume of samples of various types of cells and with the refinement of the results of multiclassification

scholarly journals Acute myeloid leukaemia: an unusual cause of biliary strictures

2019 ◽  
Vol 12 (3) ◽  
pp. e227821
Author(s):  
Adele Beck ◽  
Hannah Hunter ◽  
Simon Jackson ◽  
David Sheridan
Keyword(s):  
Bone Marrow ◽  
Obstructive Jaundice ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Blood Count ◽  
Extrahepatic Bile Duct ◽  
White Blood Count ◽  
Full Blood Count ◽  
Significant Past Medical History ◽  
Acute Myeloid

A 17-year-old man with no significant past medical history presented with a 2-week history of worsening jaundice, lethargy, anorexia and progressive right upper quadrant abdominal pain. There were no stigmata of chronic liver disease. Initial investigations were suggestive of cholangitis with large intrahepatic and extrahepatic bile duct strictures but otherwise normal hepatic and splenic appearances. A percutaneous transhepatic cholangiogram with the positioning of drains was performed to alleviate the obstructive jaundice. Within 2 weeks of the first presentation, full blood count revealed a significantly raised white blood count and a subsequent peripheral blood smear and bone marrow were consistent with a diagnosis of acute myeloid leukaemia. Chemotherapy was started after partial improvement of his obstructive jaundice. Complete morphological and cytogenetic remission was obtained 4 weeks after the first cycle of chemotherapy (half dose of daunorubicin and full dose of cytarabine, treated off trial) on control bone marrow. The patient remains in remission.

Study on Methylation of p15INK4B Gene and Its Mechanisms in Patients with Myelodysplastic Syndrome and Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3441-3441
Author(s):  
Hongyan Tong ◽  
Maofang Ling ◽  
Jie Jin
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Acute Leukemia ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Chronic Phase ◽  
Lymphocytic Leukemia ◽  
Low Risk ◽  
Blast Phase ◽  
Genetic Event

Abstract The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in the mononuclear cells (MNCs) from bone marrow of 54 cases with hematopoietic malignances were detected by using RT-PCR, Western blot, and methylation-specific PCR. Of the 54 patients, 10 cases were low-risk MDS, 10 cases were high-risk MDS, 10 cases were acute myeloid leukemia (AML), 10 cases were acute lymphocytic leukemia (ALL), 10 cases were chronic myeloid leukemia in chronic phase (CML-CP), and 4 cases were CML in blast phase (CML-BP). 10 normal persons were studied as nective controls. The results showed that the incidence of p15INK4B methylation in cells of high-risk MDS was higher than that in low-risk MDS (6/10 VS 1/10, P=0.003), and the p15INK4B methylation was found to be associated with the down-regulation of the expressions of p15INK4B gene on both mRNA (r=−0.734, p<0.001) and protein (r=−0.664, p=0.001)levels, which indicated that the silencing of p15INK4B gene was in conjunction with hypermethylation in MDS. The expressions of p15INK4B on mRNA level and protein levels were almost detected in the MNCs from bone marrow of normal persons without the p15INK4B methylation. We also found the expression of DNMT3A and DNMT3B in high-risk MDS (densitometry readings respectively: 0.624±0.146, 0.577±0.344) were higher than in low-risk MDS (densitometry readings respectively: 0.487±0.300, 0.338±0.290) (P<0.05). The expression of DNMT1 was higher in the groups of low-risk MDS, high-risk MDS, AL and CML-CP( densitometry readings respectively: 0.487±0.218, 0.697±0.243, 0.706±0.463 and 0.867±0.375) than in normal control (densitometry reading: 0.181±0.312)(P<0.05, figure listed bellow), which indicated that up-regulated DNMTS might contribute to the hypermethylation of p15INK4B, and the higher expressions of de novo methyltransferases DNMT3A and DNMT3B may be related to the disease progression of MDS. The methylation of p15INK4B was also detected in 9/20 of AL cases accompanied by over-expressions of DNMT1, DNMT3A, and DNMT3B (densitometry readings respectively: 0.706±0.463, 1.066±0.547, and 0.530±0.428). The methylation of p15INK4B was detected in 1 of 10 cases of CML-CP patients, but all be detected in 4 case of CML-BP patients. These results indicated that the hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of high-risk MDS, acute leukemia, and blast phase of CML. Furthermore, DNMT3A and DNMT3B were substantially over-expressed in the bone marrow cells of these patients. which might play an important role in the transformation from MDS to acute leukemia. Figure Figure

Automated Quantitative Assessment of Bone Marrow in Normal Samples: Correlation with the Reference Method.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4192-4192
Author(s):  
Gina Zini ◽  
Mariagrazia Garzia ◽  
Antonella Di Mario ◽  
Elena Rossi ◽  
Giuliana Farina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Count ◽  
Blood Count ◽  
Hematopoietic Cells ◽  
Reference Method ◽  
Mean Value ◽  
White Blood Count ◽  
Quantitative Detection ◽  
Microscope Examination ◽  
Age Related

Abstract Bone marrow (BM) analysis is conventionally performed by microscope examination on films of about 0.3ml of aspirated bone marrow fluid, stained with Romanowsky dyes. Until 1996 the simple automated screening of marrow composition was made very difficult by a number of factors, mainly the lack of the erythroblasts quantitation and the fat interference. From 1996 last generation automated hematology analyzers provide accurate and precise erythroblasts counts; moreover same systems have improved their software reducing the problem of fat interference. We have analyzed data from 100 normal BM samples from patients submitted for diagnostic and/or follow up purposes in our Hematology Day Hospital. BM fluid was harvested from the superior posterior iliac crest. The first 0,3–0.5 ml were used for smears, while the next 1–2 ml of BM, collected into K3-EDTA, were analysed with Coulter LH 750, a fully automated hematology analyzer which provides Complete Blood Count, White Blood Count Differential included Nucleated Red Blood Cells (NRBC) and Reticulocytes count. We used the microscope examination conventionally performed on films stained with Romanowsky dyes as reference method. Quantitative detection BM cellularity was obtained by semi quantitative evaluation based on the evaluation of hematopoietic cells in several marrow particles: physiological differences age related were also taken in account. If hematopoietic cells occupy less than 25% or more than 85% the sample is defined respectively hypocellular or hypercellular (none of our sample was as). Differential cell count was usually performed on two different slides counting 500 cells (1000 when hypercellular, but none of our sample was as). We found a strict correlation between microscope semi-quantitative cellularity evaluation and the instrumental cell count as sum of WBC plus NRBC, the Total Nucleted Cell Count (TNCC). The mean value of the TNCC in normal PM samples was 29,48 x109/L with a range 25,9–54,9 x109/L. These results are in good agreement with normal BM cell count reported in the literature using a cytofluorimetric method, which is 34,5 x109/L (SD28.0). The instrumental mean percentage of BM granulocytes corrected for TNNC was 62% (range: 23,5–93,7) versus a mean microscope percentage of 58,42% (range: 40–72). The automated NRBC BM count corrected for TNCC was 11,38% (range: 2,7 – 39,17) versus a microscopic mean value of 28% (range: 9–45). These results, including the slight NRBC underestimation probably due to partial mature cell lysis, are in line with the data of the literature. This study confirms the feasibility of routine automated cell count using a hematology in normal BM fluid samples. Automated methods will support morphologists quickly providing accurate and precise quantitative information such as TNCC and myeloid/erythroid ratio.

Correlation between WT1 Levels and Chimeric Transcripts in Core Binding Factor AML: Discrepancies Are Not Uncommon AML1-ETO Leukemias.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2309-2309
Author(s):  
Adriana Lasa ◽  
Maite Carricondo ◽  
Daniela Cilloni ◽  
Salut Brunet ◽  
Anna M. Aventin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Copy Number ◽  
Residual Disease ◽  
Wilms Tumor ◽  
Quantitative Polymerase Chain Reaction ◽  
Core Binding Factor ◽  
Acute Leukemias ◽  
Healthy Donors ◽  
Binding Factor

Abstract The Wilms tumor gene (WT1) was identified as a tumor suppressor gene coding for a zinc-finger transcription factor. It has been demonstrated that WT1 is overexpressed in acute leukemias and detection of increased levels of WT1 transcripts mainly in peripheral blood, has been associated with clonal growth and relapses. Most AML patients do not have a suitable molecular marker for minimal residual disease (MRD) monitoring. WT1 quantification seems to be an attractive strategy of universal leukemia follow-up. Bone marrow samples from 46 core binding factor (CBF) AML patients were tested for WT1 expression in parallel to AMLI/ETO and CBFb/MYH11quantification. Total RNA was purified using the Trizol reagent (Invitrogen). Chimeric detection was performed following the BIOMED recommendations. The WT-1 mRNA expression was measured by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI7700 genetic analyzer (Applied Biosystems, Foster City). For WT1 copy number titration the IPSOGEN plasmid was employed (Ipsogen, Marseilles). One hundred and fifty bone marrow (BM) samples from AML1-ETO patients (23 samples at diagnosis and 127 during follow-up) and 195 from CBFbeta-MYH11 cases (21 samples at diagnosis and 174 during follow-up) were included in the study. Bone marrow samples from 6 healthy donors were used to establish the highest acceptable value of WT1 copy number (80 copies). The WT1 expression was significantly increased (up to 3 orders) in bone marrow samples of AML patients at diagnosis compared to BM samples of healthy donors (P < 0.0001). Relapses were observed exclusively in the CBFbeta-MYH11 group and were always preceded by rising amounts of WT1 levels. A good concordance between WT1 levels (80 copies) and prognostically relevant chimeric trancript copy number (1 copy) was detected in 96.23% of the samples. Discrepancies between WT1 and specific fusion genes were observed in 11 AML1-ETO samples and in two CBFbeta-MYH11 cases. In ten follow up AML1-ETO samples with copy values of 1 or less a WT1 result exceeding the 80 threshold was detected. This discordant result was found in a single CBFbeta-MYH11 sample. Our findings suggest that WT1 is a reliable MRD marker in CBFbeta-MYH11 AML. It remains to be investigated the meaning of high titers of WT1 in AML1-ETO acute leukemias.

Novel Leukemia Associated Immunophenotype (LAIP) Absent from Normal and Regenerating Bone Marrow Identified by Multiparameter 6-Color Flow Cytometry.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2362-2362
Author(s):  
Denis Guyotat ◽  
Daniela Olaru ◽  
Pascale Flandrin ◽  
Nathalie Nadal ◽  
Lydia Campos
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Color Flow ◽  
Significant Difference ◽  
Blast Cells ◽  
Disease Study ◽  
New Generation ◽  
Minimal Residual

Abstract Flow cytometry analysis of minimal residual disease (MRD) in acute myeloid leukemia (AML) is based on the detection of aberrant phenotypes responsible for the relapse. Until now, all studies were performed by 3 or 4 color immunostaining, allowing the identification of LAIP in 80% of cases. Moreover, no data is available regarding the existence of such phenotypes in regenerating bone marrow. The new generation of cytometers allows the study of 8 parameters that permit a better distinction of malignant from normal phenotypes. In our study we analyzed 20 bone marrow samples from allogeneic donors, 20 ALL regenerating bone marrows after chemotherapy and 53 AML samples at diagnosis. Multiparameter 4 colour and 6 colour flow cytometry was used in order to define antigen combinations which are totally absent or present at very minimal levels in normal and regenerating hematopoiesis. “Blast cells” were gated according to CD45/SSC properties.For the first time we describe by 6 color flow cytometry 47 phenotypes totally absent from “blasts” gate in all normal bone marrow (ex: CD34+DR−117+33−15+, CD34+38+33−56+19−, CD14−DR+4+11B+64+). Another 41 phenotypes were identified as presents at a frequency < 0,05% of total cells (ex: CD34+DR+117−33+15+, CD14−DR+4+11B+64−, CD34+65−56+4−16−). There was no significant difference between normal and regenerating marrows. The 4 color panel of moAbs allowed us to identify only 30 phenotypes presents at a frequency < 0,05% of total cells (ex: CD34+33−13+, CD34+117+11b+, CD34+DR−13+). 53 AML at diagnosis were studied using 6 color immunophenotyping and 58 % of phenotypes described as aberrant or infrequent in normal myeloid hematopoiesis were found in at least one AML at diagnosis in more than 1% of total cells. All AML cases show at least one LAIP but frequently we observed more than one LAIP blast subpopulation in the same sample. Some examples of LAIP observed are CD34+ 38+ 33+ 56+ 19−, CD34+ 38+ 33+ 56− 19+, CD34− DR− 117+ 33+ 15−. In conclusion our results shows that (1) the ability to clearly distinguish leukemic from the healthy cells is considerably increased by 6 color approach (8 parameters analyzed) than 4 color. (2) Furthermore that these aberrant or infrequent phenotypes in normal or regenerating bone marrow samples are identified in AML cases and can be utilized in AML minimal residual disease study. (3) Knowledge of the expression of different markers in normal hematopoietic development provides a frame of reference for identification of abnormal differentiation patterns.

Sign in / Sign up

Export Citation Format

Share Document