Detection of Molecular Relapse of AML by Very Frequent Quantitative Monitoring of Different Molecular Markers (Fusion Transcripts and WT1) in Different Compartments (Peripheral Blood-PB, Bone Marrow-BM, and Selected CD34+ Bone Marrow Cells) and Its Treatment with Chemotherapy or Gemtuzumab Ozogamicin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4481-4481
Author(s):  
Jiri Mayer ◽  
Ivo Palasek ◽  
Zdenek Pospisil ◽  
Sona Struncova ◽  
Dana Dvorakova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Subsequent Development ◽  
Fusion Transcript ◽  
Gemtuzumab Ozogamicin ◽  
Time Interval ◽  
Clinical Relapse ◽  
Fusion Transcripts ◽  
True Value

Abstract Patients and Methods. In order to find some markers, which reliably enable to predict clinical relapse in AML patients, we primarily focused on patients with the known fusion transcript (CBFB-MYH, AML1-ETO, or involving the MLL gene) and correlated this values with the WT1 for estimation of the true value of WT1 monitoring of disease behavior in a given patients. The value of different compartments for minimal residual disease (MDR) monitoring (PB, BM or CD34+ BM cells) was also analyzed. The study was prospective; in the case of MDR dynamics, the patients were actively called for earlier visit. This strategy could estimate the real time interval from MRD level increase to hematological relapse detection. In the interventional part of the study, the patients with already known MRD dynamics were treated at the time of molecular relapse. Results and Discussion. In 67 AML patients and 3 healthy volunteers, 2184 BM or PB samples were examined, including 240 samples from CD34+ BM cells. Follow-up was 31–252 weeks (median: 88 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0.9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1 values were frequently >0 even if the level of fusion transcript =0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM or CD34+). Molecular relapse was defined as a reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. The time from molecular to hematological relapse was 8–79 days (median: 25 d). In the cases of subsequent development of hematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34− blasts. Eight patients were treated for 13 molecular relapses with following results: chemotherapy, CR=2, PR=2; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=2 (PR was defined as a decrease in fusion transcript level at least 10-fold). Patients with CD33− blast at diagnosis did not respond to gemtuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment. Conclusion: Fusion transcript monitoring enables reliable detection of molecular relapse in AML and high values in CD34+ BM cells signalize imminent hematological relapse (even in the case of CD34− blasts). PB is a suitable compartment for frequent monitoring. However, in some cases, relapse are fulminate, hardly allowing any intervention. WT1 does not seem to be a reliable marker for exact molecular relapse detection. AML at the stage of molecular relapse behaves similarly to AML at the frank hematological relapse: there are CRs, PRs, or NRs when using chemotherapy or gemtuzumab ozogamicin. AML with CD33− blasts at diagnosis does not seem to respond to gemtuzumab ozogamicin at the stage of molecular relapse. Success of AML therapy in the future seems to be dependant on efficient targeting the leukemia stem cell.

Detection and Treatment of Molecular Relapse in Acute Myeloid Leukemia with RUNX1, CBFB or MLL Gene Translocations. Frequent Quantitative Monitoring of Molecular Markers in Different Compartments and Correlation with WT1 Gene Expression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4356-4356
Author(s):  
Michael Doubek ◽  
Ivo Palasek ◽  
Zdenek Pospisil ◽  
Sona Struncova ◽  
Dana Dvorakova ◽  
...  
Keyword(s):  
Residual Disease ◽  
Subsequent Development ◽  
Fold Increase ◽  
Fusion Transcript ◽  
Clinical Relapse ◽  
Wt1 Gene ◽  
Fusion Transcripts ◽  
Mll Gene ◽  
Quantitative Monitoring ◽  
Acute Myeloid

Abstract Background. It seems that the population of leukemia stem cells (LSCs) have fundamental importance in the origin and maintenance of the acute myeloid leukaemia (AML). Eradication of LSCs is a new goal of AML therapy. We hypothesized, that by monitoring of minimal residual disease (MRD) and its dynamics in different compartments (peripheral blood, PB; bone marrow, BM; sorted CD34+ BM cells; and CD34- BM cells) it would be possible to find some patterns reliably predicting clinical relapse. Aims. To find which compartment is the best for MRD monitoring and whether it would be possible to treat the disease in the phase of molecular relapse in order to prevent the hematological relapse. Methods. MRD monitoring, in average once or twice per month, was performed in all phases of treatment, and was done even more frequently in the cases of unstable MRD. RQ PCR for fusion transcripts (CBFB/MYH11, RUNX1(AML1)/ETO or fusion transcripts of MLL gene) and WT1 gene was used. Molecular relapse was defined as reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. Some patients with already known MRD dynamics and high probability of imminent hematological relapse were treated at the time of molecular relapse. Results. In 67 AML patients and 6 healthy volunteers, 2352 BM or PB samples were examined, including 265 samples from CD34+ BM cells. Follow up was 31–287 weeks (median: 113 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0,9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1values did not mostly reach zero values even if the level of fusion transcript was 0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM, CD34− or CD34+). In relapsed patients, the time from molecular to haematological relapse was 8 – 79 days (median: 25 d). In the cases of subsequent development of haematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34- blasts. Nine patients were treated for 17 molecular relapses with following results: chemotherapy, CR=2, PR=3, NR=1; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=3, NR=1 (PR was defined as a decrease in fusion transcript level at least 10-fold, CR as a decrease to 0). Patients with CD33- blasts at diagnosis did not respond to gentuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment. Conclusion: Frequent quantitative monitoring (especially in CD34+ BM cells) of fusion transcripts (in contrast to WT1) is useful for reliable prediction of haematological relapse in AML patients. PB seems to be sufficient for frequent outpatient MRD monitoring. Efficient targeting of LSCs will be essential for AML cure, however, the best method is currently not known. Some now available procedures are sometimes surprisingly successful.

Use of Plasma RNA for Real-Time Quantitative RT-PCR to Monitor Imatinib Therapy in Patients with Chronic Myeloid Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1099-1099
Author(s):  
Vilmos A. Thomazy ◽  
Hagop M. Kantarjian ◽  
Maha Imam ◽  
Peter J. Davies ◽  
Christine Aguilar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Imatinib Therapy ◽  
Rt Pcr ◽  
Cytogenetic Remission ◽  
Plasma Rna

Abstract Monitoring minimal residual disease (MRD) in chronic myeloid leukemia after imatinib therapy can help in determining therapeutic strategy. Sensitive methods based on the detection of the BCR/ABL fusion gene or its fusion transcript by FISH or RT-PCR, respectively, are used to monitor residual leukemia in peripheral blood or bone marrow cells. While sensitive, these methods require an invasive procedure to obtain bone marrow. The patchy nature of the residual disease may cause some of the reported variation in detecting MRD. We hypothesized that plasma RNA level reflects total body disease and is unaffected by the tendency of some myeloid cells not to circulate. We measured the BCR/ABL fusion transcript by real-time quantitative RT-PCR using RNA isolated from blood plasma. We used a quantitative RT-PCR assay capable of measuring both the b2a2 and b3a2 BCR/ABL fusion transcripts by targeting exon 13 of BCR with the 5′ and exon 2 of ABL by the 3′ primer. RNA isolated from 10–30 μl of frozen plasma was subjected to real-time quantitative RT-PCR using the Taqman technology. Quantification was achieved by extrapolation from a standard curve generated from a ten-fold dilution series of a synthetic oligonucleotide corresponding to the (+) strand of the target sequence. To normalize the fusion mRNA levels we also measured β-actin mRNA from the same samples. Assays were run in duplicate. The results of plasma RT-PCR were correlated with results of conventional cytogenetic analysis, FISH, and quantitative RT-PCR of bone marrow suspension (“bone marrow PCR”). Plasma and bone marrow samples were obtained at 3, 6, 9, 12 and 15 months after imatinib therapy. Ninety-five plasma samples were analyzed. There was a strong positive correlation between the results of plasma RT-PCR vs. positive cytogenetics (n=95, p<0.00001), FISH (n=88, p<0.00001), and bone marrow RT-PCR (n=74, p<0.00001). At 12 months after imatinib therapy all but 2 of the 29 patients were in complete hematologic and cytogenetic remission (CR); one patient was in partial remission (PR) and one in hematologic, but not cytogenetic, remission. These two non-CR patients were positive by both bone marrow and plasma-based RT-PCR assay for the fusion transcript. Three of the four CR patients positive by both plasma and bone marrow PCR at 12 months progressed and converted to PR/minimal response at 15 months post-therapy. The level of plasma BCR/ABL fusion mRNA at 12 months was significantly higher (p=0.04) in these patients than in those who did not progress. Our results show that, by using 10–30 μl of plasma per assay, RT-PCR is at least as sensitive as cytogenetic or FISH analysis of bone marrow cells in monitoring MRD.

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

Minimal residual disease in acute lymphoblastic leukemia detected by immune selection and gene rearrangement analysis.

1989 ◽  
Vol 7 (3) ◽  
pp. 338-343 ◽  
Author(s):  
M Bregni ◽  
S Siena ◽  
A Neri ◽  
R Bassan ◽  
T Barbui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
Gene Rearrangement ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Immune Selection ◽  
Gene Rearrangement Analysis ◽  
Minimal Residual

We have developed an assay for the detection of malignant residual cells in the bone marrow from patients with B- or T-lineage acute lymphoblastic leukemia (ALL) in clinical remission. This assay involves an immune selection step followed by immunoglobulin or T-cell receptor gene rearrangement analysis and allows the detection of one contaminating tumor cell out of 1,000 normal bone marrow cells. We have examined the bone marrow of 11 patients with adult ALL in remission over a 24-month period. Five patients relapsed in the bone marrow and one in the CNS. The assay allowed the detection of minimal residual disease in four of five patients that subsequently relapsed in the bone marrow, 1.5 to 9 months before the relapse became morphologically and clinically manifest. Residual disease was not found in the bone marrow from patients in continuous remission and from the single patient who relapsed in the CNS. We conclude that the ability of the assay described here to detect minimal residual disease with high specificity can provide information for further understanding of the biology of ALL and hopefully for the clinical management of patients with this disease.

scholarly journals Detection of residual leukemic cells in patients with acute promyelocytic leukemia by the fluorescence in situ hybridization method: potential for predicting relapse

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 495-499 ◽  
Author(s):  
L Zhao ◽  
KS Chang ◽  
EH Estey ◽  
K Hayes ◽  
AB Deisseroth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Low Frequencies

Abstract The translocation between chromosomes 15 and 17, t(15;17)(q22–24;q11– 21), is present in the bone marrow cells of most patients with acute promyelocytic leukemia (APL). Although conventional cytogenetic methods are useful for diagnosing this disease, difficulties are experienced in detecting residual disease among those patients who have achieved remission. In this study, we used the fluorescence in situ hybridization (FISH) method to attempt to detect residual leukemic cells in 10 APL patients in clinical remission. The duration of remission ranged from 2 to 93 months at the time of study. Multiple bone marrow samples were analyzed by FISH in most patients. In 6 patients, no cell with t(15;17) was found. These patients remain in complete remission at present (approximately 25 to 33 months since first studied by FISH). In 4 patients, low frequencies of cells with t(15;17) were observed in at least one bone marrow sample examined. All of these patients relapsed within 1 to 14 months. No cell with t(15;17) was identified by the conventional G-banding method in any sample. The FISH results correlated well with that of a two-round nested reverse transcription polymerase chain reaction assay that was performed on the same samples. Thus, our study suggests that FISH is potentially a useful tool for detecting residual APL cells and for identifying patients at high risk of relapse.

Use of 5-Fold Staining for Multiparameter Flow Cytometry-Based Quantification of Minimal Residual Disease in Acute Myeloid Leukemia Results in Improved Sensitivity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2025-2025
Author(s):  
Wolfgang Kern ◽  
Daniela Voskova ◽  
Claudia Schoch ◽  
Wolfgang Hiddemann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Normal Bone Marrow ◽  
Prognostic Information ◽  
Normal Bone ◽  
Triple Staining ◽  
Marrow Cells

Abstract The quantification of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) using triple staining has been shown to yield prognostic information independent of other parameters in patients with acute myeloid leukemia (AML). Due to the immunophenotypic heterogeneity of AML the application of 5-fold staining may result in a better characterization of the leukemia-associated aberrant immunophenotype (LAIP) and thus in an improved sensitivity of the method as compared to triple staining. We analyzed bone marrow samples from 114 patients with newly diagnosed and untreated AML by MFC using a comprehensive antibody panel with 5-fold combinations. Sensitivity was estimated by quantification of LAIP-positive cells for each LAIP in 18 normal bone marrow samples. In each patient at least one LAIP was identified (total, 203 LAIPs). The LAIPs were present on a median of 15.88% of the bone marrow cells at diagnosis (range, 2.11% to 79.64%). The median number of normal bone marrow cells displaying the LAIPs ranged from 0.001% to 0.065% (median, 0.010%). As a result, the logarithmic difference (LD) in LAIP-positive cells between leukemic and normal bone marrow amounted to a median of 3.33 (range, 1.96 to 4.88). Similarly, if only the most sensitive LAIP was considered for each patient the median frequencies of LAIP-positive cells were 14.07% (range, 2.11% to 77.57%) in leukemic bone marrow and 0.010% (range, 0.001% to 0.065%) in normal bone marrow. Importantly, however, in this setting the resulting LD amounted to a median of 3.45 (range, 1.96 to 4.88). In order to estimate the impact of applying 5-fold staining on the sensitivity the information of each of the applied colors was skipped once while the results of the other four colors, respectively, were used. Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells (median, 0.050%; range, 0.001% to 3.6%) while the percentages of LAIP-positive leukemic cells changed only marginally (median, 22.65%; range, 2.25% to 90.06%). The gain in LD by applying 5-fold staining in comparison to 4-fold staining amounted to a median of 0.58 (maximum gain, 3.14). In 32 patients a total of 120 follow-up samples have been analyzed appyling the combination of antibodies that allowed the best LAIP definition. The LD from diagnosis to follow-up amounted to a median of 2.82 (range, 0.77 to 4.82). Clinical follow-up data is available in 26 of these 32 patients. MRD assessment after completion of consolidation therapy has been performed in 15 patients. The median LD between diagnosis and follow-up assessment is 2.84 (range, 1.07 to 4.33). Separating patients according to this median LD identified a group of patients with no relapses yet (LD >2.84) while patients with an LD <2.84 had an event-free survival of only 50% at one year (p=0.075). These data confirm that flow cytometrically-based assessment of MRD is feasible in AML and results in prognostic information. It is suggested that the application of 5-fold staining significantly improves the sensitivity and thereby the overall accuracy of the method.

Determination of Minimal Residual Disease in Patients with Acute Myeloid Leukemia by Multiparameter Flow Cytometry: Prognostic Impact at Different Checkpoints.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 400-400 ◽  
Author(s):  
Wolfgang Kern ◽  
Daniela Voskova ◽  
Claudia Schoch ◽  
Wolfgang Hiddemann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Impact ◽  
Minimal Residual ◽  
Marrow Cells

Abstract Guiding antileukemic treatment in patients with acute myeloid leukemia (AML) is increasingly based on levels of minimal residual disease (MRD) which can be quantified with high sensitivity by multiparameter flow cytometry (MFC). The optimum checkpoint for determination of MRD during the course of therapy, however, has not yet been determined. We applied MFC using a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIPs) at diagnosis and to quantify MRD by individually selected antibody combinations. The prognostic impact of MRD levels was assessed in comparison to cytogenetics and age. Patients received double induction, consolidation, and maintenance therapies and underwent allogeneic stem cell transplantation if they were younger than 60 years and had a matched related donor. In 286 patients with newly diagnosed and untreated AML MFC-based assessment for the presence of LAIP has been performed. The median percentage of LAIP-positive bone marrow cells at diagnosis was 16.04% (range, 2.54%–76.14%). All individual LAIPs were applied to 26 normal bone marrow samples to estimate sensitivity based on the median percentages of LAIP-positive normal bone marrow cells which ranged from 0.00% to 1.01% (median, 0.02%). A total of 550 follow-up samples has been analyzed in these patients at different checkpoints (CP1, up to day 21 after start of therapy, n=85; CP2, day 22–60, n=122; CP3, day 61–120, n=158; CP4, day 121–365, n=137; CP5, after day 365, n=48). In order to adjust for differences in the percentages of LAIP-positive bone marrow cells at diagnosis the logarithmic difference (LD) between diagnosis and follow-up was calculated for each follow-up sample. The median LDs at the respective checkpoints were: CP1, 2.02; CP2, 2.29; CP3, 2.39; CP4, 2.53; and CP5, 2.81. Separation of patients according to the respective median LDs resulted in differences in event-free survival (EFS; CP1: 21.1 vs. 9.1 months, p=0.0711; CP2: 14.2 vs. 9.3 months, p=0.0095; CP3: 30.9 vs. 13.5 months, p=0.0055; CP4: median not reached vs. 14.1 months, p<0.0001; CP5: median not reached vs. 22.5 months, p=0.0001) and overall survival (OS; CP3: median not reached vs. 21.6 months, p=0.0332; CP4: 90% vs. 53% at 2 years, p=0.0058). Cox analysis using the LDs at the different checkpoints as continuous variables confirmed the prognostic impact on EFS (CP2, p=0.002; CP3, p=0.0003; CP4, p<0.0001; CP5, p<0.0001) and revealed an impact also on OS (CP3, p=0.003; CP4, p=0.001; CP5, p=0.029). Cox regression analysis taking into consideration cytogenetics and age as covariates proved the independent prognostic impact of LD at checkpoints 2 to 5 on both EFS and OS with the exception of LD at checkpoint 2 and OS. In fact, LD at checkpoint 5 was the only parameter independently related to EFS and OS. These data suggest that quantification of MRD by MFC in AML results in powerful and independent prognostic parameters. In particular during the first year of treatment MRD levels provide important prognostic information. Clincal trials should use MRD-based stratification in order to assess the efficacy of early treatment intensification in high-risk AML patients.

High Frequency of Molecular Remissions (m-CR) in Multiple Myeloma Patients Achieving a Hematologic Complete Remission (CR) on Total Therapy II (TT-2).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 933-933
Author(s):  
Guido Tricot ◽  
Matteo Carrabba ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
John Shaughnessy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Detection Threshold ◽  
M Protein ◽  
Pcr Analysis ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Marrow Cells

Abstract TT-2 represents a very intensive treatment approach for newly diagnosed myeloma patients consisting of hematopoietic growth factor-dependent induction therapy (VAD, DCEP, CAD, DCEP); followed by melphalan 200 mg/m2-based tandem autotransplant; 4 quarterly consolidation chemotherapy cycles with D-PACE; and interferon alpha maintenance with added dexamethasone pulsing during the first year. A stringently defined hematologic CR (immunofixation negative, normal bone marrow aspirate and biopsy, and absence of a light-chain restricted aneuploid population of bone marrow plasma cells by DNA/cIg flow cytometry) was obtained in 48% of the first 446 patients enrolled on TT-2. A CR defined as such probably reflects a detection threshold of 108–109 tumor cells. CDR3-PCR to evaluate minimal residual disease (MRD) in myeloma has revealed m-CR in 50% of 40 reported allotransplant cases compared to approximately 15% among 75 reported autotransplants myeloma. We evaluated the frequency of m-CR in 20 TT-2 patients with a qualitative PCR for CDR3, which can detect 1 myeloma cell in 105–106 normal bone marrow cells (Corradini et al. J Clin Oncol 1999, 17: 208). CDR3 probes were generated from RNA of CD138 immunogenetic bead-purified plasma cells obtained at diagnosis. Light-density (<1.077 g/cm3) bone marrow cells served as the source of DNA post-therapy to assess MRD. The quality of DNA was determined by amplification of the p53 exon 6 sequence on all CDR3-PCR negative cases. At the time of CDR3-PCR analysis, 13 patients were in hematologic CR, 1 in near CR (immunofixation positivity as the only evidence of disease), 4 in partial remission (PR) (bone marrow < 5% plasma cells, decrease in serum M protein ≥ 75% and in urine M protein ≥ 90%) and two had less than a PR. Four patients were tested for MRD prior to the first transplant (2 < PR, 1 PR, 1 CR), two prior to the second transplant (2 PR) and 14 at a median of 21 months (range 6 months to 2 years) after the second transplant (12 CR, 1 near CR, 1 PR). Six of the 13 CR patients had abnormal metaphase cytogenetics at diagnosis, including 5 with deletion of chromosome 13. A m-CR was observed in 10/13 (77%) hematologic CR patients, including 4/6 with abnormal cytogenetics and 6/7 with normal cytogenetics. P53 exon 6 amplification was seen in all patients who were CDR3-PCR negative. Not unexpectedly, all seven patients failing to achieve a stringently defined hematologic CR had persistent disease as assessed by CDR3-PCR. Our results suggest that high intensity therapy as applied in TT-2 can indeed achieve a more marked tumor cytoreduction as reflected by a higher m-CR rate than previously reported with autotransplantation also in patients with baseline abnormal metaphase cytogenetics, and that the m-CR rate appears to be at least equivalent to that observed after allotransplantation. It remains to be shown whether achieving a m-CR will result in prolonged survival and a potential of cure in myeloma as has been shown convincingly for other hematologic malignancies. An additional 35 hematologic CR patients have samples available for CDR3-PCR analysis so that, at the time of presentation, a more definitive assessment of TT-2’s potential to effect a m-CR can be presented.

CpG Oligonucleotides Induce Anti-Leukemia Activity in a Syngeneic Murine Model of Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2830-2830
Author(s):  
Alix E. Seif ◽  
Marlo D. Bruno ◽  
Junior Hall ◽  
Valerie I. Brown ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Intensive Therapy ◽  
Significant Proportion ◽  
Cpg Odn

Abstract Acute lymphoblastic leukemia (ALL) accounts for 80% of all pediatric leukemias and is the most common form of childhood cancer. While most children with ALL are cured by current therapies, refractory and relapsed ALL comprise a significant proportion of all pediatric cancers. Furthermore, nearly half of all ALL diagnoses occur in adults, who carry a much poorer prognosis, with the majority dying of relapsed disease. Relapsed ALL generally requires intensive therapy with significant associated morbidity and mortality. Development of novel therapies is essential to improving outcomes. DNA oligodeoxynucleotides containing CpG motifs (CpG ODN) stimulate anti-tumor immune activity via Toll-like receptor 9 (TLR9) activation and are currently in clinical trials for a variety of solid tumors. We have previously reported that CpG ODN stimulation alters antigen presentation by human ALL cells, enhancing allogeneic Th1 responses. In addition, we have shown that CpG ODN administration in vivo reduces the leukemic burden of primary human ALL xenografts in Nod-SCID mice, and that this activity is mediated in part by NK cells. To further the development of CpG ODN as a novel therapeutic agent for ALL, we have investigated the induction of anti-ALL activity by CpG ODN in a syngeneic ALL setting. CpG ODN did not exhibit direct toxicity against cell lines derived from leukemic Eμ-ret transgenic mice in vitro, nor did it alter CD40 or CD86 expression or cytokine production. However, using a flow cytometry-based in vitro killing assay we observed CpG ODN-induced elimination of leukemia cells when cultured with splenocytes or bone marrow cells from Eμ-ret transgene-negative mice (P=0.0388). The difference between CpG ODN-treated and untreated controls became more pronounced with increasing effector:target ratios (P<0.0001). Preliminary data show that depletion of NK cells markedly decreases the magnitude of the observed effect, supporting the hypothesis that this cell type is involved in targeted control of ALL in this model. The ability of CpG ODN to exert anti-leukemia activity in a syngeneic setting suggests that it may have utility as an adjuvant therapy. To test this hypothesis we administered CpG ODN (or PBS) to syngeneic leukemia-bearing mice 2 days after completion of a chemotherapy regimen used to reduce leukemia burden. When mice were sacrificed 3 weeks after treatment, we found significantly reduced leukemia burden in bone marrow (P=0.0019), spleen (P<0.00001) and blood (P=0.00028) of CpG ODN-treated mice. Cell-depletion and cytokine-neutralization assays are currently ongoing to define the mechanism of action of CpG ODN in these settings. To our knowledge, this is the first demonstration of CpG ODN-induced anti-ALL activity in a post-chemotherapy syngeneic model, suggesting that this agent has the potential to treat minimal residual disease and to reduce the incidence of relapse.

Three Novel AML Cases Harboring the Semi-Cryptic t(7;21)(p22;q22) Translocation Expressing RUNX1-USP42 Fusion Transcripts Associated with Diploidy/Tetraploidy and/or 5q Alterations: a Probably Underestimated Combination

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2708-2708
Author(s):  
Eric Jeandidier ◽  
Carine Gervais ◽  
Isabelle Radford-Weiss ◽  
Catherine Gangneux ◽  
Valerie Rimelen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Fusion Transcript ◽  
Fish Analysis ◽  
Chimeric Transcript ◽  
Rt Pcr ◽  
Balanced Translocation ◽  
Fusion Transcripts ◽  
Cryptic Translocation

Abstract Abstract 2708 RUNX1 is implicated in numerous chromosomal abnormalities acquired in acute myeloid leukemia (AML). The most frequent one, the t(8;21) is associated with a particular morphology together with a favorable prognosis. This is not the case for other 21q abnormalities, that are much less frequent and for which the prognosis is quite different. Moreover, beside point mutations, conventional cytogenetics failed to detect some of chromosomal alterations involving RUNX1. Recently 3 cases of the rare and semi-cryptic t(7;21)(p22;q22) translocation expressing the RUNX1-USP42 fusion transcripts have been reported, demonstrating the recurrence of this abnormality in AML. We describe here 3 additional cases with the same translocation and fusion transcripts, associated to 5q alterations leading to EGR1 and CSF1R heterozygous losses. In all our patients, the t(7;21)(p22.1;q22.3) was initially detected by the systematic FISH evaluation of the blastic populations using ETO-AML1 Dual Fusion probe. Patient#1 bone marrow karyotype was characterized by a tetraploid clone (89,XXYY) with loss of chromosomes 15, 17 and 18 in addition to the t(7;21), and a unbalanced translocation der(5)t(5;13)(q23;q?) between long arms of chromosomes 5 and 13, resulting in a heterozygous loss of EGR1 and CSF1R. Patient #2 blood and bone marrow karyotypes revealed a diploid clone with a del(5)(q31q33) associated with the t(7;21). The FISH analysis confirmed EGR1 and CSF1R deletions. In patient #3, the bone marrow karyotype showed diploid/tetraploïd clones, both harboring the t(7;21)(p22;q22), confirmed by FISH experiments (WCP7, AML1 probes). In addition, a der(5)t(1;5)(q3?2;q21-23) was identified within the tetraploïd clone, resulting in the loss of EGR1 and CSF1R, confirmed by FISH. In all three cases a RUNX1-USP42 fusion transcript was detected using RT-PCR, as well as the reciprocal transcript. Sequence analysis of RT-PCR products showed that the breakpoints occurred exactly in the same introns of USP42 and RUNX1 as in the previously described cases. For patient #1 and #3 a chimeric transcript was found formed of the RUNX1 exon 7 fused to the USP42 exon 3. In patient #2, a shorter chimeric transcript arised from the fusion of the RUNX1 exon 5 to the exon 3 of USP42. As already noticed in the previous reports, an alternative splicing of the RUNX1 exon 6 has been detected in these three cases. The description of these 3 novel t(7;21) confirm the recurrence of this balanced translocation in AML, and shows that this chromosomal abnormality is often associated with diploid/tetraploid clones and/or 5q alterations. Special attention should be paid in karyotype analysis of AML with diploid or tetraploid clones harboring 5q alterations. In such cases RUNX1 rearrangements should be explored using FISH analysis, and RUNX1-USP42 fusion transcript should be searched by RT-PCR in positive cases. Prospective and retrospective studies of AML have now to be settled in order to assess the incidence and clinical relevance of this cryptic translocation. Disclosures: No relevant conflicts of interest to declare.

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