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.

Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

Pretreatment Patient Characteristics Associated with Achieving Bone Marrow Minimal Residual Disease-Free Status with Frontline Fludarabine, Cyclosphosphamide, Rituximab (FCR) Chemoimmunotherapy for CLL,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3902-3902
Author(s):  
William G. Wierda ◽  
Susan O'Brien ◽  
Alessandra Ferrajoli ◽  
Charles Asa Koller ◽  
Jan A. Burger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Odds Ratio ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Color Flow ◽  
End Of Treatment ◽  
Free Status ◽  
Minimal Residual

Abstract Abstract 3902 Chemoimmunotherapy (CIT) is highly effective treatment and standard of care for patients (pts) with CLL. Response to treatment by NCI-WG/IWCLL criteria correlates with outcome; pts who achieve complete remission (CR) have superior progression-free and overall survival compared to pts who achieve partial remission (PR); and pts who fail therapy have the poorest outcome. Emerging data indicate improved outcomes for pts who achieve minimal residual disease (MRD)-free status in blood or bone marrow (BM) by end of treatment. We are conducting a clinical trial to prospectively evaluate pretreatment pt characteristics and prognostic factors and correlations with NCI-WG response, MRD-free status, and time to event outcomes with standard frontline fludarabine, cyclophosphamide, and rituximab (FCR) CIT. A total of 197 pts have been registered, 160 have completed treatment and are evaluable for response by NCI-WG criteria, and 127 have BM MRD status evaluated by standard 4-color flow cytometry at Course 3 and/or end of treatment. We report on pretreatment characteristics associated with MRD-free status at end of treatment. For the 160 pts evaluable for response by NCI-WG criteria, 63% were male; the median (range) age, β2M, and absolute lymphocyte count (ALC) were 58 yrs (38–84), 3.6 mg/l (1.3–14.1), and 78.7 K/μl (.8–394), respectively. The percent pts with Rai high-risk disease, unmutated IGHV status, ZAP70+ by immunohistochemistry (IHC) and CD38+ (30% cutoff) was 35%, 60%, 63%, and 37%, respectively. According to the hierarchical categorization, FISH demonstrated 17p del in 9%, 11q del in 18%, +12 in 17%, 13q del in 36%, and no abnormality in 20% of pts. The median number of FCR courses given was 6; 57% received all intended 6, 21% received 4–5, and 23% received ≤3. Of the 160 pts, 63% achieved CR, 12% nodular PR (nPR), 23% PR and 3% did not respond. Of 127 pts with BM evaluated by 4-color flow cytometry at end of treatment, 56% were MRD-free. Of 71 MRD-free pts, 27 were negative at end of course 3, 33 converted to negative after course 3, and 11 were negative at end of treatment but did not have a course 3 evaluation. Univariable Chi-square analyses demonstrated pretreatment β2M, IGHV mutation status, 17p del, and +12 correlated with MRD-free status at end of treatment (Table). The following did not correlate: age, Rai stage, WBC, ALC, HGB, PLT, ZAP70, CD38, or number of FCR courses received. Multivariable logistic regression model identified β2M≥4 mg/l (odds ratio=.78; p=.007) and unmutated IGHV (odds ratio=.77; p=.006) as independently associated with lower likelihood to achieve MRD-free status. In conclusion, mutated IGHV and β2M <4 mg/l are independently associated with increased likelihood of achieving MRD-free status with frontline FCR CIT; further follow up is needed to correlate MRD-free status with improved survival outcomes for patients treated on this trial.TableNCI-WG Responsen% MRD-NegativeCR8071nPR150PR3047*NR20Pretreatment Characteristicn% MRD-Negativep-valueAge (yrs) <65100600.07≥652741Rai Stage Low & Int-risk82610.12High-risk4347b2M (mg/l) <473640.02≥44942ALC (K/ml) <5040560.86≥508755IGHV Mutated47700.006Unmutated6244ZAP70 IHC Negative41610.28Positive7351CD38+ ≤7%4863Ref**8–29%27480.23≥30%42550.46FISH 13q del4556RefNone22680.32+1220800.0611q del20400.2417p del10200.04*All MRD-free are PR due to cytopenia, with no evidence of CLL**Used as reference or comparison group Disclosures: No relevant conflicts of interest to declare.

Carfilzomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma: Final Results from the NCI Phase 2 Pilot Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4746-4746 ◽  
Author(s):  
Ola Landgren ◽  
Mark Roschewski ◽  
Sham Mailankody ◽  
Mary Kwok ◽  
Elisabet E. Manasanch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Color Flow ◽  
Smoldering Multiple Myeloma ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract BACKGROUND: Early treatment with lenalidomide and dexamethasone delays progression and increases overall survival in patients with high-risk smoldering multiple myeloma. The addition of the selective proteasome inhibitor carfilzomib to a lenalidomide and dexamethasone backbone has proven effective in patients with newly-diagnosed multiple myeloma; this combination may allow patients with high-risk smoldering multiple myeloma to obtain deep and durable responses. METHODS: In this phase 2 pilot study, patients with high-risk smoldering multiple myeloma received eight 28-day cycles of induction therapy with carfilzomib (at a dose of 20/36 mg per square meter on days 1, 2, 8, 9, 15, and 16), lenalidomide (at a dose of 25 mg on days 1–21), and dexamethasone (at a dose of 10 or 20 mg on days 1, 2, 8, 9, 15, 16, 22, and 23). Patients achieving stable disease or better after combination therapy received 2 years of maintenance therapy with lenalidomide. Minimal residual disease was assessed with multi-color flow cytometry, next-generation sequencing by the LymphoSIGHT method, and fluorodeoxyglucose-positron emission tomography-computed tomography (FDG-PET/CT). Myeloma clonotypes were identified in genomic DNA obtained from CD138+ bone marrow cell lysate or cell-free bone marrow aspirate at baseline for each patient based on their high frequency within the B-cell repertoire. Per study protocol, minimal residual disease assessment by next-generation sequencing, multi-color flow cytometry and FDG-PET/CT was repeated when patients achieved a complete response or completed 8 cycles of induction treatment. A sample size of 12 evaluable patients was calculated as being minimally necessary based on the following probability calculations: If the true probability of a very good partial response was 20% or 50%, we calculated that there would be a 7.3% or 80.6% probability, respectively, if 5 or more patients exhibiting a very good partial response (VGPR). Thus, if 5 or more patients out of 12 achieved a very good partial response, there would be strong evidence that the true probability of a VGPR was 50% or more. RESULTS: Twelve patients were enrolled. All 11 patients (100%) who completed 8 cycles of combination therapy obtained VGPR or better (primary end point). Minimal residual disease assessment by next-generation sequencing was performed on bone marrow supernatant to detect cell-free myeloma clonotypes, while flow cytometry analysis utilized bone marrow cells. Overall (N=12), 100% of patients achieved a complete response or better over the study period, including 11 patients (92%) negative for minimal residual disease based on multi-color flow cytometry. Based on next-generation sequencing, two of the 12 patients were positive for minimal residual disease in the bone marrow supernatant; one of these two patients was also positive for minimal residual disease based on multi-color flow cytometry in the bone marrow cells. Information regarding longitudinal minimal residual disease status will be available and presented at the meeting. Adverse events were manageable. CONCLUSIONS: Early treatment with carfilzomib, lenalidomide, and dexamethasone was associated with high rates of complete response and minimal residual disease negativity by multi-color flow cytometry, next-generation sequencing, and FDG-PET/CT in patients with high-risk smoldering multiple myeloma. Disclosures Landgren: Onyx Pharmaceuticals: Consultancy; Medscape: Consultancy; Millennium Pharmaceuticals: Independent Data Monitoring Committee (IDMC), Independent Data Monitoring Committee (IDMC) Other. Off Label Use: Carfilzomib and lenalidomide for high-risk smoldering multiple myeloma.

scholarly journals Established Standards of Minimal Residual Disease Detected By Multiparametric Flow Cytometry in Acute Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5262-5262
Author(s):  
Hui Jing ◽  
Fen Huang ◽  
Zhengshan Yi ◽  
Zhongxin Zheng ◽  
Xiaolei Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Remission Induction ◽  
Test Results ◽  
Color Flow ◽  
Multiparametric Flow Cytometry ◽  
Minimal Residual

Abstract Purpose To establish a method for detecting minimal residual disease (MRD) by eight color flow cytometry which is stable, repeatable and accurate quantitation. Method According to the ratio of 10%, 1%, 0.1%, 0.01% and 0.001%,to analyze sensitivity of the method by successively mixing the cell lines (Kasumi, KG-1a) and primary acute leukemic bone marrow cell were mixed with normal bone marrow cells. In order to ensure the specificity of the test results, we increased antibody number to eight color combinations of antibodies to adjust fluorescence compensation value after labeling antibody separately in each channel. To verify the feasibility of standardization, standardized test were in 25 bone marrow samples of acute leukemia. Result In standard conditions of detection and sensitivity of 10-5could be detected by eight color flow cytometry. In our study there were 25 cases of acute leukemia, including 14 patients with acute myeloid leukemia and 11 patients with acute B-lymphoblastic leukemia. 23 of 25 cases were detected specific leukemia associated immunophenotypes (LAIP) at diagnosis. 20 patients could be detected the original LAIP, and LAIP of 3 patients changed after remission induction therapy. To analyze the relationship between the clinical data and MRD level, the result showed that the type of LAIP had significant influence on level of MRD. After remission the level of MRD in patients who expressed cross lineage and non-synchronous antigens at diagnosis was significantly higher than those who did not express (P=0.003, P=0.006). Conclusion We established the standardized conditions of minimal residual disease detected by multiparametric flow cytometry to ensure the accuracy and specificity of the test results. It has important significance to confirm that manifestations of LAIP were consistent with the outcome in patients with acute leukemia. Disclosures No relevant conflicts of interest to declare.

Plasma Alemtuzumab Levels at End of Treatment Correlate with Response and Response Duration in Patients with CLL Receiving Treatment for Residual Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2109-2109
Author(s):  
William G Wierda ◽  
Thomas J Kipps ◽  
Michael J Keating ◽  
Jennifer R Brown ◽  
John G Gribben ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Response Duration ◽  
Response To Treatment ◽  
Color Flow ◽  
Median Response ◽  
End Of Treatment ◽  
Minimal Residual

Abstract We conducted a phase II trial with alemtuzumab self-administered, subcutaneously (SQ) to eliminate residual disease after chemotherapy or chemoimmunotherapy for patients (pts) who were in partial remission (PR) or pts in complete remission (CR) with 2-color flow cytometry evidence of disease. Pts received alemtuzumab SQ 3mg, 10mg, 30mg on days 1,2,3 then 30mg thrice weekly for a total of 12 doses, including rampup. After the 12th dose, pts with residual disease could receive a second course of 12 additional doses. Responding pts were pts in PR who converted to nodular PR (NPR) or CR by NCI-WG ‘96 criteria or pts in CR who remained in CR and had no evidence of disease by 2-color flow cytometry. There were 26 pts enrolled in PR, 5 pts in CR. 7 pts received a second course of alemtuzumab. End of treatment plasma alemtuzumab levels were measured by a flow cytometry-based assay (Rebello and Hale, J Immunol Methods, 2002). The response rate in the intent-to-treat population was 74%. We retrospectively evaluated patients’ bone marrow for minimal residual disease by 4-color flow cytometry. A disappointing 18% of pts evaluated was MRD-negative in bone marrow by this method. Responding pts had mean plasma alemtuzumab level of 8μg/ml verses 3μg/ml for non-responders (p=.003). The median plasma alemtuzumab level for responders was 6μg/ml. Responders with end of treatment plasma alemtuzumab level greater than 6μg/ml had a significantly longer median response duration of 21.2 months versus 8.9 months for pts with less than 6μg/ml (p=.048). End of treatment plasma alemtuzumab level correlated with response to treatment and response duration for patients treated with alemtuzumab SQ. Future studies to optimize dose and schedule for alemtuzumab SQ as treatment for CLL, including for residual disease, should include measurement of end of treatment plasma alemtuzumab levels and evaluation of bone marrow for minimal residual disease by 4-color flow cytometry. Furthermore, consideration of these results should be given to optimize the effective duration of treatment with alemtuzumab SQ.

Single Immunophenotypical Evaluation of Minimal Residual Disease before Autotransplantation of Non-Cryopreserved Bone Marrow Is Insufficient for Prediction of Outcome in High Risk Adult Acute Lymphoblastic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4439-4439
Author(s):  
Beata M. Stella-Holowiecka ◽  
Krystyna Jagoda ◽  
Aleksandra M. Holowiecka-Goral ◽  
Tomasz Czerw ◽  
Sebastian Giebel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Conditioning Regimen ◽  
Long Term Results ◽  
Color Flow ◽  
Childhood All ◽  
Minimal Residual

Abstract For high-risk adult ALL patients alloHCT is a preferable option. However, a significant proportion of those not having a suitable donor may be successfully treated with autotransplantation (autoHCT). Based on our experience this treatment ensures low transplant related mortality below 3% and a reasonable overall survival and disease free survival of 60% and 45% respectively. The status of the disease before transplantation is an important factor for long term results. In childhood ALL most studies suggest that the level of minimal residual disease (MRD) after induction evaluated immunophenotypically or with bio-molecular methods is predictive for outcome after different treatments including chemotherapy, alloHCT and autoHCT. The results in adult ALL are more controversial. Patients selection. Among 1205 haematopoetic cell transplantations performed in our institution 224 (147 autologous, 77 allogeneic) were performed in 205 adults with ALL. For this study we selected an uniform group of 81 patients fulfilling following criteria’s: Ph (-) ALL, status CR1, evaluable MRD, strictly defined autoBMT procedure performed until the end of 2003. Methods. MRD was tested before autoBMT (median interval 10 days) using 2 ore 3-color flow-cytometry, as appropriate. The atypical immunophenotypes were evaluated using the “quadrans” analysis in all cases and since 2002 also the “empty spaces” technique. The sensitivity equals at least 0.0001. For all autoHSCT bone marrow was used as a source of stem cells. The CAV conditioning regimen consisted of cyclophosphamide 60mg/kg on d. -3, -2, cytarabine 2 g/m2 d. -3, -2, -1, etoposide 800 mg/m2 d. -3, -2. Bone marrow was not cryo-preserved after collection but stored in 40 C and re-transplanted after 72h. Results. In 41 patients; age med. 26 y (15–53), F/M=12/29, the MRD level was &lt;0,001: the MRD (−) group. In 40 patients; age med. 29 y (16–53), F/M=18/22, the MRD was detected at the level =/&gt; 0,001; MRD+ group. The ALL-immunophenotypes of MRD−/MRD+ groups were as follows; proB 4/7, preB 2/6, Common 18/19, B 0/1, preT 5/2, T 12/1). The interval from DGN to BMT was similar in both groups. The probability of LFS and OS at 10y calculated with median follow up time of 5y equaled; in the MRD(−) group 47% and 62% and in the MRD+ one 48% and 57% respectively (p=ns). The main reason of failure in both groups was a relapse which occurred after a median time of 277 days in the MRD(−) group and 134 days in MRD+ one (p=0.19). Conclusion and comment. Based on this observation we conclude that a single evaluation stratifying patients before autoBMT according to MRD level below or above 0.001 is not predictive for DFS and OS, because it informs only about the current amount of the disease but not about its opportunistic nature. In this respect a repeatedly confirmed MRD positivity should be more significant. Taking into consideration that the main reason of failures were relapses, this finding suggests also that in patients with chemotherapy-responsive ALL confirmed by stabile CR, the myeloablative CAV regimen is sufficiently strong to eliminate the residual disease at the level ranging 0.01–0.001. It may be speculated only that the 72h lasting incubation of bone marrow product before re-transplantation has also some kind of purging effect for leukemic blasts.

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.

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