Using CD160 as a Novel Approach to the Assessment of Minimal Residual Disease (MRD) in Chronic Lymphocytic Leukaemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2036-2036
Author(s):  
Timothy Farren ◽  
Jerome Giustiniani ◽  
Ulrika Johansson ◽  
Christina Lim ◽  
Gemma Athorn ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Magnetic Beads ◽  
Nk Cell ◽  
Residual Disease ◽  
Cytotoxic T Cells ◽  
Becton Dickinson ◽  
Flow Cytometric ◽  
Colour Flow ◽  
Low Levels

Abstract Despite recent therapeutic advances, B-cell CLL (CLL) remains an incurable disease. Achieving MRD negativity in CLL has been associated with improved outcomes. Historically, co-expression of CD5 and CD19 with kappa/lambda light-chain (LC) restriction has been the mainstay of evaluating MRD in CLL. Using both 2 and 4-colour flow cytometry, interpretation was still complicated by the presence of normal CD5+ B cells and the difficultly of establishing LC restriction, as well as its significance, at very low levels of B-cells. CD160 is an activatory NK cell receptor also found on a subset of cytotoxic T-cells. CD160 is not expressed on normal B-lymphocytes or myeloid cells. We have previously reported that CD160 is aberrantly expressed on malignant B-cells, including >98% of CLL cases1,2. Using anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France), we have developed a highly sensitive multi-colour, single tube flow cytometric (MC-ST-FC) assay. Whole blood (1×106 leukocytes) were labelled with antibodies, followed by a lysed/washed approach using an ammonium chloride-based lysing and fixing methodology, and analysed on a FACS Canto (Becton Dickinson). In excess of 10,000 events were recorded within the ‘disease gate’ and MRD assessed by CD19+/CD160+/CD5+/CD2-/CD3- co-expression was compared with CD5+/CD19+ co-expression and LC restricted analysis. Dilution experiments showed the CD160 MC-ST-FC assay had a sensitivity of at least 104. PCR for CD160 mRNA was performed on highly purified B-cells (>98% CD19+ using magnetic beads). 98 samples from 36 patients were assessed by two and multi-colour flow cytometry at specified time points throughout their treatment, using a cut-off limit of 1% for CD19/CD160 and 10% for CD5/CD19 co-expression. 30 blood samples were analysed for CD160 expression using PCR. LC restriction could not be reliably measured in patients with very low levels of B-cells. Of 55 samples with <10% CD5/CD19 co-expression, 41 samples did not show LC restriction. 31 of these cases had low level CD19/CD160 expression (2 to 10%) indicating MRD positivity - these cases would otherwise have erroneously been called MRD negative. In 21 cases, <10% CD5/CD19 expression with LC analysis (6 with normal κ/λ, 8 with insufficient cells) and CD19/CD160 (with 7 cases of equivocal 1% CD19/CD160 co-expression) gave MRD negative results. These were subsequently confirmed with PCR. The remaining 37 samples showed evidence of residual disease using both >10% CD5/CD19 and >2% CD19/CD160 expression, again confirmed by PCR. Qualitative PCR results were in direct concordance with the flow assay. There were no samples in which CLL cells were detected by PCR, but not by the CD160 MC-ST-FC assay. CD160 MC-ST-FC assay is a sensitive assay for CLL MRD, with better performance than dual CD5/CD19 with LC analysis. CD160 expression is stable during, and post-therapy and is applicable to vitually all cases of CLL. There was excellent correlation with PCR. Furthermore, the CD160 MC-ST-FC assay is simpler, faster, cheaper and applicable to more patients, than current molecular and flow cytometric methods. Agrawal S. et al. Blood, 100 (Abstract no. 1488) 2002 Agrawal S. et al. Blood, 94 (suppl 1), p119a, 1999

Monitoring of T-cell acute lymphoblastic leukemia by flow cytometry

Open Medicine ◽  
2010 ◽  
Vol 5 (6) ◽  
pp. 651-658
Author(s):  
Miglė Janeliūnienė ◽  
Rėda Matuzevičienė ◽  
Laimonas Griškevičius ◽  
Zita Kučinskienė
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric ◽  
Analysis Strategy ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Positive Results ◽  
Color Marker

AbstractMinimal residual disease (MRD) predicts the outcome of acute lymphoblastic leukemia (ALL). Flow cytometry (FC) is one of the most sensitive and most applicable methods for MRD diagnostics, but there is still no agreement on the “gold standard” of the method. We tried to optimize flow cytometric MRD detection in T-ALL. Fourteen adults and 11 children with T-ALL and 12 normal bone marrow (BM) donors were enrolled in the study. We found that the most common phenotypic aberrations in T-ALL were TdT and CD99 coexpression on T-cells in BM. Therefore for MRD detection we developed a limited four-color marker panel (TdT/CD7/cCD3/CD19 and CD99/CD7/cCD3/CD2) and a standard analysis strategy. This assay was evaluated on BM of healthy controls. Less than 0.01% TdT+ or CD99 bright T-cells were found in normal BM. MRD was detected in 9 adult patients and 1 child at different time-points of treatment. The average TdT and CD99 mean fluorescence intensity (MFI) value of residual blasts fluctuated during therapy, but it still remained higher than MFI of normal T-cells. Our established MRD detection method differentiated leukemic lymphoblasts with sensitivity in the range of 0.01% and did not give any false positive results in normal BM.

A Single-Tube Seven-Colour Flow Cytometry Assay for Detection of Minimal Residual Disease In Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1688-1688
Author(s):  
Soraya Wuilleme ◽  
Nelly Robillard ◽  
Steven Richebourg ◽  
Marion Eveillard ◽  
Laurence Lodé ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Flow Cytometry Assay ◽  
Single Tube ◽  
Cell Identification ◽  
Colour Flow ◽  
Multiparameter Analysis ◽  
Minimal Residual

Abstract Abstract 1688 The eradication of minimal residual disease (MRD) in myeloma predicts for improved outcome. A number of different approaches to myeloma MRD detection are available; these vary widely in sensitivity and cost. Flow cytometric assessment of MRD may be preferable in practice because of lower cost and easier feasibility. Myeloma MRD flow cytometry requires at least three markers for plasma cell identification (CD38, CD138 and CD45) and combination of several additional markers to detect phenotypic abnormality including CD19, CD20, CD27, CD28, CD45, CD56 and CD117. Also, assessment of immunoglobulin light-chain restriction (cytoplasmic K and L) combined with myeloma-associated phenotypic plasma cell abnormalities, is very important. Four-tube four-colour flow cytometry combine markers CD38/CD138/CD45 with markers for plasma cell phenotypic abnormalities and clonality. Six –colour flow cytometry combines the same markers (markers for plasma cell identification) plus clonality markers; it potentially increases the sensitivity of the method through coincident multiparameter analysis. However, the single-tube six-colour flow cytometry, proposed by others studies, excludes the myeloma-associated phenotypic plasma cell abnormalities and consequently decreases specificity of the assay. We propose a new single-tube seven-colour flow cytometry, including plasma cell identification antigens, clonality markers and myeloma-associated phenotypic plasma cell abnormalities markers. In this new method, PCs are stained with antibodies: (i) CD38, CD138, CD45 used for identified plasma cells and percentage plasma cells to total leucocytes. (ii) CD19 and CD56+CD28 used to identify normal and abnormal plasma cells; and (iii) cy-IgK and cy-IgL, for confirm the plasma cells clonality. We analysed normal bone marrow provided from healthy individuals. Our results showed a presence myeloma-associated phenotypic plasma cell abnormalities at low levels in healthy individual. The monotypy studies confirm polyclonality of this normal plasma cells. Then we compared MRD assessement with single-six colour flow cytometry assay (plasma cells markers, clonality markers and exluding myeloma-associated phenotypic markers) and seven-colour flow cytometry assay (including myeloma-associated phenotypic markers). Six –colour flow cytometry has a better sensitivity and showed efficacy for quantification MRD in myeloma patients. However, the single-tube six-colour flow cytometry excluded the myeloma-associated phenotypic plasma cell abnormalities and in some cases the seven-colour flow cytometry will be more informative because it detected myeloma-asociated phenotypic marquers combined with clonality marquers. Finally, the single-tube seven colour flow cytometry assay provides reduction in antibody cost and increases sensitivity and specificity of the method through coincident multiparameter analysis. Disclosures: No relevant conflicts of interest to declare.

Augmentation of Sensitivity of FLT3/ITD Assay Allows Detection of Minimal Residual Disease In Stem Cell Transplant Recipients-Correlation with Flow Cytometric MRD Assessment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1717-1717
Author(s):  
Maya Danielle Hughes ◽  
Rong Zeng ◽  
Kristen L. Miller ◽  
Soheil Meshinchi
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Flow Cytometric ◽  
Detection Assay ◽  
Minimal Residual

Abstract Abstract 1717 FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutation that is associated with therapy resistance in acute myeloid leukemia (AML). Early data demonstrated low sensitivity for this assay, thus limiting its utility to the evaluation of diagnostic specimens, and precluding its utility in remission samples. We inquired whether the standard FLT3/ITD assay can be modified to enable its utility to detect presence of residual disease in remission specimens. Enhanced FLT3/ITD assay sensitivity was accomplished by altering annealing temperature, increasing the number of cycles as well as amount and concentration of the product that was subjected to capillary electropheresis. To assess the sensitivity of the enhanced assay, FLT3/ITD positive cells M4V11 were serially diluted in a population of ITD negative cells (HL60). The concentration of M4V11 cells in each sample ranged from 10% to 0.0001%. PCR product was subjected to capillary electropheresis and the appropriate region of the electropherogram was examined for the presence of the appropriate mutant product length. Appropriate FLT3/ITD signal was detected in dilutions down to 0.01%, validating our ability to detect extremely low levels of FLT3/ITD. We subsequently examined the remission marrows from patients with a history of FLT3/ITD who had undergone stem cell transplantation. Available bone marrow specimens (N = 51) from patients who underwent stem cell transplantation for FLT3/ITD-positive AML were analyzed and the result was correlated with the available standard PCR as well as the available MRD assessment by muti-dimensional flow cytometry; samples negative for FLT3/ITD by standard assay (N=11) were then subjected to the enhanced PCR methodology. Available ITD length for each patient was used for examination of the appropriate region of the electropherogram in each case. Of the available 51 bone marrow specimens analyzed, 23 specimens had FLT3/ITD detectable by standard PCR protocol. Using our modified PCR method and capillary electrophoresis, an additional 13 specimens had identifiable FLT3/ITD. In 6/11 patients, where initial FLT3/ITD was negative by standard methodology, enhanced assay identified FLT3/ITD signal. In each case, detection of FLT3/ITD by the enhanced assay was followed by morphologic or immunophenotypic emergence of disease, prompting therapeutic intervention. We further evaluated the ability to detect FLT3/ITD in patients with minimal residual disease by flow cytometry. 33 of the bone marrow specimens analyzed had a less than 5% abnormal blast population as detectable via flow cytometry. Among these samples, 7 had FLT3/ITD detectable using standard detection techniques. An additional 11 samples had detectable FLT3/ITD when our modified protocol was employed. Of the specimens that had less than 1% abnormal blast population as detectable via flow cytometry (N = 27), 4 had FLT3/ITD detectable using the standard detection assay; when our modified protocol was employed, an additional 6 samples had detectable FLT3/ITD. 17 bone marrow specimens had no abnormal blast cells detectable via flow cytometry; of these samples 1 had detectable FLT3/ITD using the standard detection assay, while an additional 3 had detectable FLT3/ITD using our modified assay. In four patients, FLT3/ITD was detected in bone marrow specimens found to have flow cytometric MRD of 0% (N=2), 0.1% (N=1) and 0.4% (N=1). In two patients with no detectable disease by MDF, both had emergence of morphologic (60% blast) or immunophenotypic disease by MDF (1.1%) within 4–6 weeks of detection of FLT3/ITD by enhanced assay. In this study, we demonstrate that simple modifications to the FLT3/ITD genotyping assay significantly increases its sensitivity and provides a highly sensitive and very specific assay for identifying this disease associated mutation in remission specimens. The enhanced assay can be incorporated into the standard evaluation of remission status for patients with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

High-Throughput Screening by Flow Cytometry Identifies Reduced Expression of CD48 As a Universal Characteristic of T-ALL and a Suitable Target for Minimal Residual Disease (MRD) Detection

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2547-2547 ◽  
Author(s):  
Brent L. Wood ◽  
Greg Levin ◽  
Megan Wilson ◽  
Stuart S. Winter ◽  
Kimberly Dunsmore ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Differential Expression ◽  
Fluorescence Intensity ◽  
Pediatric Patients ◽  
Nk Cell ◽  
Residual Disease ◽  
Median Fluorescence Intensity ◽  
Flow Cytometric

Abstract Abstract 2547 The flow cytometric detection of minimal residual disease (MRD) relies on the differential expression of antigens between normal and leukemic cell populations of similar lineage. In T-cell lymphoblastic leukemia (T-ALL), the principal normal populations from which leukemic blasts must be distinguished are mature T cells and NK cells, as immature T cells are not normally present in peripheral blood or bone marrow. Current methods rely on a relatively small number of antigens, some of which are not stable following therapy. In particular, immature antigens such as TdT and CD99 that distinguish leukemic and mature cells at diagnosis often revert to mature levels after therapy. The subset of T-ALL cases having expression of surface CD3 can be particularly problematic. Consequently, the identification of novel antigens to supplement those currently in use is highly desirable. We undertook a systematic search for novel antigens capable of distinguishing T-ALL from mature T cell and NK cell populations using a high-throughput flow cytometric screening technique. LyoPlates (Becton-Dickinson) containing antibodies against 242 unique antigenic specificities in a 96 well plate format were used to assay mononuclear cells obtained from 3 normal peripheral blood donors and 9 pediatric patients with T-ALL. The T-ALL cases covered the range of immunophenotypes seen in this disorder. The 9-color assay (1 detection and 8 gating fluorochromes) was performed on an LSRII and was capable of identifying discrete T cell, NK cell and Blast cell subpopulations. Comparison of the median fluorescence intensity of each of the 242 unique antigens identified CD48 as one of the few antigens that showed consistent differential expression between mature T cells and NK cells in comparison to leukemic blasts from T-ALL. To determine whether differential expression of CD48 represents a universal characteristic of the disorder, we assayed 126 consecutive pretreatment T-ALL samples from pediatric patients enrolled on Children's Oncology Group protocol AALL0434. An 8 color flow cytometric assay was employed using the following antibody combination: CD48 FITC, CD5 PE, CD16+CD56 PE-Cy5, CD3 PE-Cy7, CD8 V450 or BV421, CD4 A594, CD7 APC, CD45 APC-Cy7. Comparison of the median fluorescence intensity of CD48 between leukemic blasts and normal T and NK cells within the same specimen revealed consistently decreased expression of CD48 on leukemic blasts (see Figure 1) with some variation in intensity. This finding was highly statistically significant (p<4E-105). The stability of CD48 expression following therapy was determined by assay of CD48 expression in 126 bone marrow samples obtained at day 29 following induction chemotherapy, representing paired samples from the patients whose pretreatment samples were assayed above. The same 8 color reagent combination was used in conjunction with the standard MRD assayed utilized for the treatment protocol, a two-tube 9 color assay. Of these samples, 50 (39.7%) showed detectable MRD using the standard assay. At end induction, the ratio of CD48 median fluorescence intensity between the leukemic MRD population and normal T and NK cells within the same sample remained abnormal in most cases. It was unchanged in 26% of cases, decreased (i.e. became more aberrant) in 24%, and increased in 50%, but in most of the latter cases remained significantly <1. However, in 5 cases (10%), CD48 increased to the level seen on normal mature T and NK cells. The changes following therapy were due largely to changes in the expression of CD48 on leukemic blasts as the level of expression of normal T and NK cells was more stable. No association between apparent immunophenotypic maturational stage or surface CD3 expression was identified. We conclude that the expression of CD48 is consistently reduced on leukemic blasts from patients with pediatric T-ALL in comparison to normal mature T and NK cells at the time of diagnosis. Following therapy, the expression of CD48 undergoes modulation, but remains different from mature T and NK cells in 90% of patients. This suggests that CD48 is a useful addition to reagent combinations for the purpose of MRD assessment in pediatric T-ALL. Disclosures: Wood: BD Biosciences: Research Funding. Borowitz:BD Biosciences: Research Funding.

Post-Induction-BM Status Combining Minimal Residual Disease, Return to MDS and Hematogones in Predicting Prognosis in Adult AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3532-3532
Author(s):  
Sung-Chao Chu ◽  
Tso-Fu Wang ◽  
Yu-Chieh Su ◽  
Ruey-Ho Kao ◽  
Yi-Fung Wu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Prognostic Impact ◽  
Post Induction ◽  
Myeloid Progenitor ◽  
Flow Cytometric ◽  
Myeloid Progenitor Cells ◽  
Minimal Residual

Abstract Abstract 3532 Introduction: The study is to analyze the prognostic impact of post-induction-BM status combining minimal residual disease (MRD), neutrophil/monocyte maturation return to MDS and quantitation of hematogones in adult AML achieving first morphologic complete remission (mCR). The hypothesis is that the detection of aberrant myeloid progenitor cells, aberrant neutrophil/monocyte maturation and hematogones in post-induction BM by flow cytometry may predict the outcome of AML in mCR even without diagnostic specimen. The positive MRD and the aberrant neutrophil/monocyte maturation will predict worse prognosis. In contrast, positive hematogones will predict better prognosis. Methods: Multidimensional flow cytometry was performed on bone marrow specimens from 47 consecutive non-M3 AML patients who had achieved mCR after standard 3+7 induction treatment. The in-remission immunophenotypic evaluation of BM was done before first consolidation treatment. The aberrant myeloid progenitor cells and aberrant neutrophil/monocyte maturation were defined by the flow cytometric scoring system (FCSS)1. Two investigators were blinded to corresponding pathologic, clinical or diagnostic flow cytometric data during initial analysis phase. After reaching agreement of the three parameters: aberrant myeloid progenitor cells (MRD ≥ 0.2% or not), aberrant neutrophil/monocyte maturation (FCSS ≥ 2 points or not) and hematogones (stages I / II B lymphoid progenitor cells ≥ 0.02% or not), those prognostic impacts on leukemia free survival (LFS) and overall survival (OS) were analyzed retrospectively. Results: Table 1 summarizes the clinical characteristics of patients according to the status of MRD and hematogones. Nine (19%) patients who had MRD ≥0.2% had a significantly worse median LFS (9.0 months vs not reached; P =.006) and worse OS (24.0 months vs not reached; P =.059). Fourteen (30%) patients who had hematogone levels ≥ 0.02% had a significantly better median LFS (not reached vs 13.5 months; P =.045) and a trend of better OS (not reached vs 24.0 months; P =.070). Six (13%) patients who had FCSS ≥ 2 points had a worse median LFS (8.0 vs 48.0 months; P =.052) but not significantly worse OS (17.0 vs 28.0 months; P =.804). A Spearman coefficient for MRD ≥0.2% and hematogone levels ≥ 0.02% was -0.317 (P <.029), indicating a mildly negative correlation. However, MRD ≥0.2% and hematogones ≥ 0.02% were almost mutually exclusive. For clinical convenience, a MRD/Hematogone score was proposed that patient with MRD ≥0.2% was defined as 1 point and hematogone ≥ 0.02% was defined as -1 point. All patients could be categorized into three subgroups: 14(30%) patients with -1 point, 24(51%) patients with 0 point and 9(19%) with 1 point. Patients were stratified based on MRD/Hematogone score to assess survival using the Kaplan-Meier method and the Log-rank test. The median LFS were not reached, 48 months and 9 months for patients with -1 point, 0 point and 1 point, respectively (P =.015; Fig. 1). The median OS were not reached (-1 point), not reached (0 point) and 24 months (1 point) (P =.088). Multivariate analyses using Cox proportional hazard model demonstrated MRD/Hematogone score was an independent predictor of LFS (HR = 2.5, 95% CI, 1.2–5.1, P =.016; Table 2) after adjusting for MRC cytogenetic classification, WBC count (above or below 50000/uL) and whether undergoing allogenetic hematopoietic stem cell transplant before 1st relapse or not. Conclusion: Even without a diagnostic specimen, flow cytometry can assess and quantify aberrant myeloid progenitor cells, aberrant neutrophil/monocyte maturation and hematogones in post-induction marrow. MRD ≥0.2% or neutrophil/monocyte abnormalities (FCSS ≥ 2) predicts shorter LFS. Hematogones ≥ 0.02% predicts longer LFS and correlates negatively with MRD ≥0.2%. Proposed MRD/Hematogone score for post-induction adult AML in mCR maybe offer a simple and practical risk assessment. And the score is worthy to be validated by prospective and larger scale study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Phase I Study of Allogeneic Natural Killer Cell Therapy Generated from Cord Blood Hematopoietic Stem and Progenitor Cells in Elderly Acute Myeloid Leukemia Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1357-1357 ◽  
Author(s):  
Harry Dolstra ◽  
Mieke W.H. Roeven ◽  
Jan Spanholtz ◽  
Basav Hangalapura ◽  
Marleen Tordoir ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Therapy ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Residual Disease ◽  
Standard Chemotherapy ◽  
Donor Chimerism ◽  
Elderly Aml

Abstract Introduction Elderly acute myeloid leukemia (AML) patients have a poor prognosis due to high relapse rates following standard therapy. Natural Killer (NK) cell alloreactivity has found to control relapse in AML in the HLA-mismatched haploidentical allogeneic stem cell transplantation (allo-SCT) setting. Moreover, allogeneic NK cell infusions can induce complete remission (CR) inpatients with advanced AML. As a consequence, adoptive NK cell transfer may be a promising treatment for elderly AML patients, who are not eligible for allo-SCT. Most clinical studies exploited NK cell products enriched from leukapheresis of haploidentical donors containing low numbers of T cells that could have contributed to the observed therapeutic effects and potentially induced graft-versus-host disease (GVHD). Therefore, we have developed a GMP-compliant culture system for the generation of large batches of NK cells from umbilical cord blood (UCB)-derived CD34+ progenitor cells, without T cell contamination. Here, we report results of a phase I dose escalation study (Dutch Trial Register nr. NTR2818) to evaluate the feasibility, safety and toxicity of allogeneic UCB-NK cell infusion following an immunosuppressive preparative regimen in elderly AML patients. Secondary endpoints were NK cell lifespan and the effects on minimal residual disease (MRD). Methods Elderly AML patients not eligible for allo-SCT, and in morphologic CR after standard therapy, were given preparative chemotherapy consisting of Cyclophosphamide (Cy;900 mg/m2/day) and Fludarabine (Flu;30 mg/m2/day) on days -6 to -2. At day 0, UCB-NK cells at a dose of 3, 10 or up to 30x106/kg body weight were infused without IL-2 treatment to study if in vivo expansion could be obtained without IL-2 support. Patients were assessed for toxicity and GVHD. Donor chimerism was measured by Q-PCR for discriminating DNA polymorphisms. NK cell expansion and phenotype were analyzed by flow cytometry. MRD was evaluated by flow cytometry and molecular techniques. Results Twelve AML patients (68-76 years) have been included, all in morphologic CR after 2 to 3 standard chemotherapy courses (n=6), or 1 standard chemotherapy course followed by subsequent treatment with hypomethylating agents (azacitidine or decitabine) (n=6). Patients were treated with Cy/Flu and an escalating dose of partially HLA-matched UCB-NK cells. Four patients had good/intermediate risk, 4 poor risk and 4 very poor risk AML. To date, 9 patients received NK cell products containing a median of 74% highly activated CD56+ NK cells, with <1x104/kg CD3+ T cells and <3x105/kg CD19+ B cells. Remaining non-NK cells were CD14+ and/or CD15+ monocytic and myelocytic cells. Follow up did not show GVHD or toxicity attributed to the NK cells. As expected, preparative Cy/Flu induced a neutropenic period of 20 ± 16 days, but no severe infections were seen. A temporary repopulation and persistence of UCB-NK cells could be detected in peripheral blood between days 1 and 8 post-infusion, which was associated with increased IL-15 plasma levels observed in most patients. Interestingly, donor chimerism increased with higher doses of infused UCB-NK cells, and donor chimerism up to 3.5% was found in bone marrow (BM) at day 7/8. Further UCB-NK cell maturation in vivo was observed by acquisition of CD16 and KIRs, while expression of activating receptors was sustained. Of the 9 treated patients so far, 5 (56%) are still in CR after 43, 35, 31, 5 and 4 months, whereas 4 patients relapsed after 5, 6 (2 pts) and 15 months. Despite morphologic CR during azacitidine treatment, residual disease of 6-7% with a leukemia-associated phenotype could be detected by flow cytometry before NK cell infusion in BM of two patients. In both patients MRD was reduced to less than 0.05% at 90 days after UCB-NK cell therapy following Flu/Cy conditioning. Conclusion These results show that GMP-compliant UCB-NK cell products containing up to 30x106 NK cells/kg body weight can be safely infused in non-transplant eligible AML patients following immunosuppressive chemotherapy. After infusion, UCB-NK cells repopulate, mature and migrate to BM without supporting IL-2 infusion. Since we observed reduction in MRD in patients on treatment with hypomethylating agents, this UCB-NK cell therapy may induce or sustain CR in elderly AML patients, and could serve as an alternative consolidation therapy for patients with refractory AML or provide bridge to allo-SCT. Disclosures Spanholtz: Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment. Bohme:Glycostem Therapeutics: Employment. Kok:Glycostem Therapeutics: Employment.

Flow Cytometric Analysis of Lymphomas: Current Status and Usefulness

2006 ◽  
Vol 130 (12) ◽  
pp. 1850-1858
Author(s):  
Zahid Kaleem
Keyword(s):  
Flow Cytometry ◽  
Hodgkin Lymphoma ◽  
Flow Cytometric Analysis ◽  
Molecular Diagnostic ◽  
Routine Practice ◽  
Becton Dickinson ◽  
Color Flow ◽  
Non Hodgkin Lymphoma ◽  
Flow Cytometric ◽  
Diagnosis And Classification

Abstract Context.—Immunophenotyping has become a routine practice in the diagnosis and classification of most cases of non-Hodgkin lymphoma, and flow cytometry is often the method of choice in many laboratories. The role that flow cytometry plays, however, extends beyond just diagnosis and classification. Objective.—To review and evaluate the current roles of flow cytometry in non-Hodgkin lymphoma, to compare it with immunohistochemistry, and to discuss its potential future applications in the molecular diagnostic era. Data Sources.—The information contained herein is derived from peer-reviewed articles on the subject published in the English-language medical literature during the years 1980 to 2005 that were identified using PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi, 1980–2005) search, various books and other sources on flow cytometry, and the author's personal experience of more than 10 years with flow cytometric analysis of lymphomas and leukemia using Becton-Dickinson (San Jose, Calif) and Beckman-Coulter (Miami, Fla) flow cytometers. Study Selection.—Studies were selected based on adequate material and methods, statistically significant results, and adequate clinical follow-up. Data Extraction.—The data from various sources were compared when the methods used were the same or similar and appropriate controls were included. Most of the studies employed 2-color, 3-color, or 4-color flow cytometers with antibodies from Becton-Dickinson, Beckman-Coulter, or DakoCytomation (Carpinteria, Calif). Results were evaluated from studies utilizing the same or similar techniques and flow cytometers. Only objective data analyses from relevant and useful publications were included for reporting and discussion. Data Synthesis.—Flow cytometry serves a variety of roles in the field of lymphoma/leukemia including rapid diagnosis, proper classification, staging, minimal residual disease detection, central nervous system lymphoma detection, evaluation of prognostic markers, detection of target molecules for therapies, ploidy analysis of lymphoma cell DNA, and evaluation of multidrug-resistance markers. It offers many advantages in comparison to immunohistochemistry for the same roles and provides uses that are either not possible or not preferable by immunohistochemistry such as multiparameter evaluation of single cells and detection of clonality in T cells. Conclusions.—By virtue of its ability to evaluate not only surface but also cytoplasmic and nuclear antigens, flow cytometry continues to enjoy widespread use in various capacities in lymphoma evaluation and treatment. Additional roles for flow cytometry are likely to be invented in the future and should provide distinctive uses in the molecular era.

Elevation of Normal B Cells in Children with ALL Can Predict Relapse.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4445-4445
Author(s):  
Joseph Kapelushnik ◽  
Itai Levi ◽  
Tikva Yermoau ◽  
Asher Moser ◽  
Atamma Haj-Yhia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Acute Leukemia ◽  
Reference Values ◽  
Predictive Marker ◽  
Multiparametric Flow Cytometry ◽  
Flow Cytometric ◽  
Immature B Cells ◽  
B Lineage ◽  
Relapsed Disease

Abstract Flow cytomerty analysis of bone marrow (BM) is performed routinely in children with acute leukemia in different phases of chemotherapy. The aim of flow cytometric monitoring is to measure the amount of residual blasts and to evaluate the efficacy of chemotherapy. Multiparametric flow cytometry may be also used to evaluate normal hematopoisis in BM of patients with acute leukemia in different phases of chemotherapy. The aim of this study was to study normal lymphopoiesis in patients with relapsed disease. Twenty-two pediatric patients with ALL were enrolled in the protocol of study. Patients were treated in accordance with BFM-98 protocol. Eighteen children were in complete remission during follow-up period, while 4 children relapsed. Three of the relapsed children had B-lineage ALL, and one had T-cell ALL. Using multiparamertic flow cytometry we measured normal pro-B cells (CD19+CD34+), immature B (CD19+CD10+CD20+ and CD19+CD10−CD20+) and mature B (CD19+CD10−CD20+) cells in BM of relapsed patients in different phases of chemotherapy. Amounts or different subsets of B cells were compared with reference values determined in patients with relapse-free disease. Reference values were expressed as mean ± confidence interval for each subset of B cells and for each phase of chemotherapy. Elevation of immature normal B-cells was determined in all 4 patients who relapsed.Three during chemotherapy. Increased amount of mature B cells was determined 3 mo before the diagnosis of relapse in the patient who relapsed after the end of chemotherapy.This event was not observed in patient who did not relapse We conclude that elevation of normal immature B cells in BM of patient with ALL can be a predictive marker for eventual relapse.

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