Leukemia-associated Aberrant Immunophenotype Research Articles

Leukemia-associated aberrant immunophenotype: A flow cytometry-based experience of 110 cases from a tertiary care center in Northern India.

Leukemic cells express a characteristic set of "cluster of differentiation" (CD) markers, which forms the basis of the current WHO classification. Leukemia-associated aberrant immunophenotype (LAIP) refers to expression of unusual CD markers by leukemic cells, which are not normally expressed by their respective lineage. The incidence of LAIP varies considerably, and its clinical implications, prognostic relevance, and sensitivity to therapy are still debatable. This study was conducted to identify the immunophenotypic aberrancies in newly diagnosed leukemias in our Institute. This was an observational study, which included newly diagnosed leukemias on flow cytometry. Aberrant immunophenotypic expressions were recorded whenever present and were correlated with prognostic factors like age, gender, and total leucocyte count (TLC). The study included 110 newly diagnosed cases of leukemias (85 acute and 25 chronic) over 1.5 years. Immunophenotypic aberrancies were detected in 40.4% of the cases. The highest incidence of aberrations was detected in acute myeloid leukemia (60.7%). LAIPs were detected in 50% of T-acute lymphoblastic leukemia and 25% cases of in B-cell acute lymphoblastic leukemia (B-ALL). Aberrant CD33 and CD56 expression in B-ALL correlated with poor prognostic factors like higher age and higher TLC, respectively. Immunophenotypic aberrancies were present in 28% cases of chronic lymphocytic leukemia. The results of this study have generated valuable baseline data on the incidence of LAIPs in this region. This information is vital because establishing LAIPs at the time of diagnosis is crucial for disease monitoring. Some LAIPs are associated with underlying cytogenetic abnormalities and hence impact the management and prognosis.

Uncovering the Enemy: Single-Cell Transcriptional Profiling of Measurable Residual Disease (MRD) Cells

Albeit novel treatment concepts have entered the therapeutic landscape of AML, relapse rates remain high. This is attributed to treatment-refractory leukemic cells, which can be quantified by flow cytometry and molecular techniques (Measurable Residual Disease, MRD). MRD positivity is a significant prognostic biomarker for relapse and low overall survival rates. Post-remission therapy for the elimination of MRD remains a major challenge. We previously confirmed the prognostic value of early flow MRD assessment during aplasia in patients who achieve complete remission after intensive induction therapy (Koehnke et al., 2019). Here, we hypothesized that single-cell transcriptional profiling of refractory leukemic cells using flow cytometry (flow MRD) during aplasia will help to understand resistance mechanisms to therapy and to identify suitable targets for cellular immunotherapy. Flow MRD detection is based on the definition and characterization of leukemia-associated aberrant immunophenotypes (LAIPs) at the time of initial diagnosis, as described previously (Koehnke et al., 2015). A similar approach was applied to define LAIP+ cells at the time of aplasia (d16-d21 after initiation of induction therapy). For single-cell RNA-seq, LAIP+ cells from paired initial diagnosis and aplasia were sorted and subjected to transcriptional profiling using the mcSCRB-seq (molecular crowding single-cell RNA barcoding and sequencing) protocol (Bagnoli et al., 2018). We included 7 AML patients of different ELN genetic risk group (favorable, n=1; intermediate, n=4; adverse, n=2). In total, 854 LAIP+ cells from paired initial diagnosis-aplasia samples were projected using uniform manifold approximation and projection (UMAP) visualizations. We observed a distinct clustering of LAIP+ cells per patient and time point, revealing high inter-patient heterogeneity (Figure 1A). To identify common transcriptional changes across patients, we performed differential gene expression analysis of LAIP+ cells, comparing aplasia to initial diagnosis. We identified 93 genes significantly upregulated and 41 genes significantly downregulated (p < 0.01, FC >1 or <-1; Figure 1B). Among markedly upregulated genes across patients at the time of aplasia, we found 3 genes related to leukemic cell maintenance. Interestingly, SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, known to be essential for the proliferation and survival of AML cells (Buscarlet et al., 2014; Shi et al., 2013), showed a 1.99-fold increase in expression (p < 0.00). In addition, TRAP1, a candidate target in cancers with antiapoptotic function, was upregulated 1.5-fold (p < 0.00). And lastly, LIN28B, the cancer stem cell and leukemia driver gene, was also found to be upregulated by 0.7-fold in aplasia LAIP+ cells (p < 0.00). In addition, several immune-related genes including DC marker gene, LRP1 (CD91; p < 0.00, FC = 1.3) and the neutrophil/GMP- specific gene, cathepsin G (CTSG; p < 0.01, FC = 0.5) showed a significant up-regulation in aplasia LAIP+ cells. The latter was previously identified as a leukemia-associated antigen and a potential target for immunotherapy (Alatrash et al., 2017). Strikingly, LAIP+ cells at the time of aplasia harbored a LSC gene expression pattern in a patient-specific manner. Lastly, cell type prediction using the annotated dataset (van Galen et al., 2019) assigned the phenotype of LAIP+ cells mainly to GMP-like and cDC-like cells in variable abundance across patients. In summary, our data showed high inter-patient heterogeneity and phenotypic differences, making general statements on chemo-resistance challenging. However, despite this heterogeneity, LAIP+ cells at the time of aplasia harbor remarkable proliferation and survival advantage (stem cell-like phenotype), specifically, due to the upregulation of SMARCA4, TRAP1, and LIN28B genes. This phenotype is accompanied by patient-specific enrichment of LSC signature and upregulation of immune-related genes. Our data open up the path to better understand chemo-resistance and to identify targets that need to be addressed to eradicate MRD and improve relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Critical evaluation of the utility of pre- and post-therapy immunophenotypes in assessment of measurable residual disease in B-ALL.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the "Leukemia Associated Aberrant Immunophenotype (LAIP)" and "Difference from Normal (DFN)" approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

A Real-world Perspective of CD123 Expression in Acute Leukemia as Promising Biomarker to Predict Treatment Outcome in B-ALL and AML

IntroductionCD123 is overexpressed in many hematologic malignancies and found to be useful in characterizing leukemic blasts of both acute myeloid leukemia (AML) and B-acute lymphoblastic leukemia (B-ALL). CD123 has been recently found to be a marker of leukemic stem cells, and its utility to measure residual disease and potential role in disease relapse is under evaluation. Materials and MethodsHerein, we have evaluated the expression of CD123 in 757 samples of acute leukemia including 479 treatment-naive and 278 follow-up samples and compared with post-induction morphologic complete remission and measurable residual disease (MRD) status. Multiparametric flow cytometry was used for assessment of CD123 expression and immunophenotypic characterization of leukemic blasts at diagnostic and MRD assessment time points. ResultsUsing variable cutoffs of 5%, 10%, and 20% to define a case as CD123-positive, expression of CD123 was observed in 75.6%, 66.2%, and 50% of AML and 88.6%, 81.8%, and 75% of B-ALL, respectively. Of 11 patients, 7 (63.63%) had mixed phenotype acute leukemia, but none of the 12 patients with T-acute lymphoblastic leukemia showed positivity for CD123. CD123 expression at diagnosis was associated with post-induction MRD-positive status in both B-ALL (P < .001) and AML (P = .001). We also evaluated the utility of CD123 as a leukemia-associated aberrant immunophenotype and found it to be useful in both patients with AML (baseline, 50.6%; follow-up, 53%) and B-ALL (baseline, 75%; follow-up, 73.07%). ConclusionsIn conclusion, CD123 may be considered as a cardinal marker for residual disease assessment and response evaluation in AML and B-ALL.

Next Generation Flow for Multiple Myeloma Minimal Residual Disease: Igh Rearrangement NGS Is Complement to the NGF

Background: Detection of leukemia-associated aberrant immuno-phenotype is used to assess minimal residual disease (MRD) by multi-parameter flow cytometry (MFC). However, detection of MRD by MFC remains to be a challenging due to the possible change in aberrant immunophenotype during disease progress. In our present study, we compared International Myeloma Working group (IMW) treatment response and NGF MRD, including BM PC% and cytogenetics. Thereon, we conducted IgH rearrangement study by NGS in cases showing discrepant results.Methods: A total of 35 BM (35 myeloma patients at follow-up) was enrolled. We performed NGF using 8-color antibody panel using Navios flow cytometer and Infinicyt. Linearity of NGF was validated with myeloma cell line (U266) and BM specimen at initial diagnosis in myeloma patient. IgH rearrangement NGS was performed using Immunoseq assay (Adaptive Biotechnologies, Seattle, WA, USA). Paired specimen at initial diagnosis BM and follow-up BM were subjected to NGS study.Results: Detection sensitivity of NGF was <0.001%. Patients who achieved CR or sCR showed MRD negativity in 63.6% (7/11). Twenty-three patients showed neoplastic PCs above LLOQ and their response criteria were 1 sCR, 3 CR, 2 VGPR, 3 PR, 1 MR, 5 SD, 3 progressive disease, 3 relapse, and 2 with unavailable response. Four patients who did not achieve CR (1 VGPR, 1 PR, 1 MR, and 1 SD) showed MRD negativity by NGF. In 4 patients with discrepancy between IMW treatment response and NGF, we compared the results of IgH NGS at initial BM with those after treatment. NGS revealed a persistence of residual clone in 1 patient and an acquisition of new clone after treatment. One patient had same dominant clone both initial diagnosis BM (95.2%; proportion of clone) and follow-up BM (45.8%). The other patient had newly appeared clones in follow-up BM (6.12%, 5.63%, 3.42%, 3.11%, 3.09%) which clones were absent in initial diagnosis BM. The other 2 patients showed heterogeneous clones without dominant clone at follow-up BM by NGS. Results of FISH and immunofixation are summarized in Table 1. This results show IgH rearrangement NGS can detect malignant clone that could not be identified by using NGF.Conclusions: Thirty-six percent of patients who did not achieve CR showed NGF MRD negativity and NGS revealed residual clones in half of them. Switching of immunophenotypes of neoplastic PC can escape monitoring of NGF, and complementary NGS test is needed to catch such drifting clones for monitoring of MRD in MM. DisclosuresNo relevant conflicts of interest to declare.

PPROACHES TO THE ASSESSMENT OF MINIMAL RESIDUAL DISEASE IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIAS IN CONDITIONS OF TARGET THERAPY

Background. Flow cytometry (FC) algorithms of detection of minimal residual disease (MRD) are well standardized, and approximate to molecular biologic methods. However, besides informative leukemia-associated aberrant immunophenotype, which are selected taking into account a tumor phenotype at diagnostics stage, it is necessary to consider specificity of the provided taget therapy and its influence on a cell. Objective: to offer stable combinations of antigens to identify B-cell precursors in patients on therapy of blinatumomab. Materials and methods. Clinical observation of patient G. 4 years old with B-cell precursors acute lymphoblastic leukemia (ALL) (pre-pre-B immunosubtype), whom after 3 bloks of reinduction therapy, taking into account MRD-positive status, blinatumomab was appointed as a monotherapy. Tumor immunophenotype was characterized in details by FC protocol according to EuroFlow in debute and relapse of the disease. MRD monitoring was provided by 8-color FC taking into account personalized leukemia-associated aberrant immunophenotypes. Results. In patient with B-cell precursors ALL received blinatumomab, the strategy of MRD monitoring was changed. Due to the lack of CD19 expression, identification of B-cell precursors was based on expression of cyCD22 in combination with nuclear TdT and CD10. Conclusion. In case of blinatumomab’s appointment during B-cell precursors ALL therapy, it is necessary to change the strategy of B-cell precursors identification, due to the lack of CD19 expression. Detection of B-cell precursors should be provided by assessment of other pan-B lineage antigens. First of all, it is cyCD22 or cyCD79a in combination with nuclear TdT and CD10, within the limits of nucleated cells of the sample.

Use of CD 123 Expression on Blasts from AML, ALL and RAEB As Minimal Residual Disease Marker

Introduction: In recent studies,interleukin-3 receptor alpha chain (IL-3Rα or CD123) has been described as over-expressed on leukemia progenitor/stem cells and low or absent on normal hematopoietic stem cells suggesting that IL-3Rα could represent an important target for the development of new anti-leukemic drugs and a minimal residual disease (MRD) marker. However, CD123 expression on blast cells and its prognostic value in different hematologic malignancies as acute myeloid leukemia (AML), acute lymphoid leukemia (ALL) and refractory anemia with excess blasts (RAEB) are still controversial. In the present study, we retrospectively analyzed the CD123 and other lineage infidelity markers (LIM) expression on blast cells in bone marrow or blood samples from patients with AML, ALL or RAEB and the disease evolution including overall survival of these patients. CD123 was also measured on B-precursors.Methods: Ninety-seven patients suffering from hematologic malignancies including69 AMLs, 21 ALLs (18 B-ALLs and 3 T-ALLs) and 7 RAEBs were retrospectively analyzed in regard to CD123 (clone-FITC 763, BD Pharmingen) and other LIM expression measured by flow cytometry (FCM) on myeloid and lymphoid blasts from 2011 to 2014. CD123 was also analyzed on patients with hematogones (n=23). FCM was performed on 5 and 10-colour flow cytometers with extensive panels to precise leukemia-associated aberrant immunophenotype (LAIP) and blast populations were identified by CD45 vs. side scatter characteristics. The positivity of markers was considered at a threshold of 20%.Results: Among the 97 patients (including 45 deceased, 42 alive and 10 lost in follow-up patients), 54 (55.7%) had a CD123 over-expressed (including 21 deceased, 26 alive and 7 lost in follow-up patients). Forty-five patients with AML (n=69) had a CD123 over-expressed (65.2%). Among these 45 patients, 6 were lost in follow-up and 25 were in refractory/relapse disease or dead. Six patients, including 5/18 B-ALL (27.8%) and 1/3 T-ALL (33.3%), with ALL (n=21) had a CD123 over-expressed (28.6%). Among these 6 patients, 5 were in refractory/relapse disease or dead. Three patients with RAEB (n=7) had a CD123 over-expressed (42.86%). Only over-expressing CD123 patients with RAEB are still alive among the 7 RAEBs. The mean value (+/- SD) of the %CD123 on CD45low in all and in the CD123 positive cases of AML, ALL and RAEB was respectively 35.1 +/- 30.5% and 51.4 +/- 25.5%,18.1+/- 31.1% and 61.2 +/- 27.3% , 29.9 +/- 30.7% and 60.1 +/- 20.5%. CD123 over-expression was not observed on B-precursors. Among the 56 patients undergoing long-term follow-up, 10 shifts (17.9%) and 3 inversed shifts (5.4%) were observed in the CD123 expression. Among all, AML, ALL and RAEB patients, respectively 41 (42.3%), 36 (52.2%), 3 (14.3%) and 2 (28.6%) had LIM with respectively 27 (65.8%), 23 (63.8%), 2 (66.7%) and 2 (100%) patients with CD123 over-expression. Only 14.4%, 18.8%, 4.7% and 0% of respectively all, AML, ALL and RAEB patients demonstrated LIM without CD123 over-expression.Conclusions: Our observations confirm that IL-3Rα is very frequently expressed on leukemia blast cells, in about 2/3 of AMLs and probably in 1/3 of ALLs with a rare loss of expression with antigenic shift of the disease. As expected, CD123 is not expressed on hematogones. CD123 seems actually represent one of the most robust markers to follow MRD with FCM, in AML, ALL and RAEB. No significant association betweenCD123 expression and poor overall survival is clearly observed although CD123 expression seems associated with high relapse rate in ALL. Further prospective investigations are needed to assess the real impact of CD123 expression on disease-free and overall survival. DisclosuresNo relevant conflicts of interest to declare.

Quantitative Monitoring By RT-PCR of Molecular Markers on Day +100 after Allogeneic Stem Cell Transplantation Predicts Outcome in Patients with Acute Myeloid Leukemia

Introduction:Monitoring of minimal residual disease (MRD) is commonly used after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML) patients. Flow cytometry of leukemia-associated aberrant immunophenotypes (LAIP) and chimerism analyses are frequently used tools to monitor MRD. These methods have lower sensitivities and lower relapse detection rates in contrast to molecular markers monitored by quantitative RT-PCR. However, their clinical implications are still limited and only little evidence of single molecular markers is available. We report on a retrospective study of 144 patients with molecular MRD markers transplanted at the Ludwig-Maximilans-University Hospital of Munich-Grosshadern, and analyzed the prognostic values of molecular MRD Monitoring after SCT, baseline risk factors, and follow-up (FU) markers (e.g. chimerism analyses and LAIP).Patients and Methods:Between January 2000 and August 2012, 495 AML patients underwent SCT at our institution. 164 patients had molecular MRD markers and 144 patients had at least one sample with quantitative results prior and/or after SCT, and were included into the analyses. At transplantation, patients had a median age of 48 years (range, 18 -73), and 49 patients were in complete remission, while 95 patients were in a more advanced status of their disease (>CR2). 106 patients received SCT from a HLA matched (10/10) and 38 patients from a HLA mismatched donor. Grafts were obtained either from related (n=60) or unrelated (n=84) donors. Most patients received reduced intensity conditioning (n=142). In 338 bone marrow samples MRD was monitored prior to SCT, on day +30 and on day +100. During the FU period after day +100, MRD was monitored at individual intervals in 429 peripheral blood samples. Quantitative RT-PCR was performed for NPM1 mutation (n=52), MLL-PTD (n=31), RUNX1-RUNX1T1 (n=12), CBFß-MYH11 (n=14), MLL rearrangements (n=20), MDS-EVI1/EVI1 (n=10), and DEK-CAN (n=5). Sensitivities of the different RT-PCRs assays ranged between 10-4 and 10-6.Results:After a median FU of 41 months (range, 4 – 115), 43 patients (30%) relapsed. The MRD levels monitored by RT-PCR prior to SCT and on day +30 after SCT showed no significant impact on relapse-free survival (RFS) and overall survival (OS). At day +100 after SCT, MRD positivity was strongly associated with worse RFS (HR 3.1, p=0.001) and OS (HR 3.2, p=0.004). This was also reflected in a cumulative two-year incidence of relapse of 61% for MRD positive patients versus 15% for MRD negative patients (p<0.0001). In addition, a change to MRD positivity during the FU period was strongly associated with a worse RFS (Mantel-Byar, p<0.0001). Furthermore, in univariate regression models we analyzed SCT baseline factors and FU markers. The remission status prior to SCT, the NPM1 mutation status, a HLA matched donor, the platelet count at day +100 (cut-off <50 G/l), and the European LeukemiaNet risk stratification (favorable versus others) were prognostic relevant risk factors. In the multivariate model only MRD positivity on day +100 (HR 3.7, p=0.013) and a low platelet count on day +100 (HR 5.0, p=0.005) were significantly associated with worse RFS. LAIP analyses (3-colors flow-cytometry, cut-off 0.1%) and a change in chimerism (STR-PCR and XY-FISH, cut-off 80% and 90%) of sorted (CD3+) and unsorted cells at the respective time points had no influence on RFS and OS.Conclusions:Molecular MRD monitoring after SCT might be a useful tool to identify AML patients at high risk for relapse, in contrast to other FU markers, e.g. chimerism analyses and LAIP. In particular, MRD positivity on day +100 after SCT and the switch to MRD positivity during the FU period were significantly associated with worse RFS. As perspective, the individual molecular MRD course might be used as prognostic factor for the guidance of treatment post SCT. Patients with MRD positivity on day +100 after SCT or with a switch to MRD positivity in the FU period may be considered for donor lymphocyte infusions (DLI) or chemotherapeutic interventions, such as hypomethylating agents. DisclosuresSubklewe:AMGEN Inc.: Research Funding.

Combination of Complement-Dependent Cytotoxicity and Relative Fluorescent Quantification of HLA Length Polymorphisms Facilitates the Detection of a Loss of Heterozygosity.

Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.

Leukemia‐associated aberrant immunophenotype in patients with acute myeloid leukemia: changes at refractory disease or first relapse and clinicopathological findings

Multiparameter flow cytometry (MFC) is commonly used to detect minimal residual disease (MRD) during the course of chemotherapy or relapse. Only one study addressed the immunophenotypic changes in refractory disease. We studied changes in leukemia-associated aberrant immunophenotype (LAIP) in patients with refractory and relapsed acute myeloid leukemia (AML). We analyzed 47 patients (refractory=22; relapsed=25) by MFC, morphology, and cytogenetic studies. Thirty-five patients (74%) showed variably changed LAIPs. The frequently altered LAIPs were lack of lineage-specific antigen and lineage infidelity. The most frequently changed marker was CD13, followed by CD33, CD56, CD7, CD4, and CD11b. Cytogenetic clonal evolution at persistence/relapse was observed in 15 patients (32%). Morphologically, three patients (6%) showed significant changes at relapse. Patients with refractory AML had a higher association with poor cytogenetic risk and classification of AML with myelodysplasia-related changes. Positive MRD at postinduction was of prognostic significance. Allogeneic stem cell transplant improved overall survival. LAIP alterations in refractory/relapsed AMLs are common findings. Presence of persistent disease indicates a poor prognosis, regardless of cytogenetic risk or expression of CD7 or CD56. Discordance between cytogenetic and LAIP changes suggests that gross cytogenetic clonal evolution during disease progression only partly contributes to immunophenotypic instability.

The percentage of peripheral blood blasts on day 7 of induction chemotherapy predicts response to therapy and survival in patients with acute myeloid leukemia

Background Rapid clearance of peripheral blood blasts (PBBs) predicts complete remission (CR) and survival in patients with acute myeloid leukemia (AML). We aimed to explore the correlation between induction therapy response, outcome, and the PBB percentage. Methods Forty-six consecutive patients with de novo AML (excluding acute promyelocytic leukemia) were enrolled in this study. Flow cytometry was performed to identify cells with a leukemia-associated aberrant immunophenotype in the initial bone marrow aspirate and in peripheral blood on day 7 of induction therapy. Results The PBB percentage on day 7 (D7PBBP) was significantly lower in patients who achieved CR (0.03% (0.0%, 0.45%)) than in those who did not (10.85% (1.13%, 19.38%); u =-3.92, P &lt;0.001). The CR rate was significantly higher among patients with a D7PBBP of &lt;0.945% (84.62%, 22/26) than among those with a D7PBBP of ≥0.945% (25.0%, 5/20; χ 2 = 16.571, P &lt;0.001). D7PBBP was significantly correlated with overall survival (OS; r=-0.437, P=0.003) and relapse-free survival (RFS; r=-0.388, P=0.007). OS and RFS were significantly higher in patients with a D7PBBP of &lt;0.43% than in those with a D7PBBP of ≥0.43% (P &lt;0.001 and P=0.039, respectively). D7PBBP was also found to be an independent prognostic indicator in multivariate analysis for both OS (P=0.036) and RFS (P=0.035). Conclusion D7PBBP may be an important risk factor for the achievement of complete remission, for overall survival, and for relapse-free survival.

Early detection of MRD in peripheral blood after induction chemotherapy of newly diagnosed patients with AML and its correlation with curative effects

The purpose of this study was to detect the minimal residual disease (MRD) in peripheral blood of newly diagnosed patients with acute myeloid leukemia (AML) on day 8 of induction chemotherapy and analyze the correlation between day 8 MRD (D8RD) and therapeutic effectiveness. 29 adult patients (13 males and 16 females, aged 16 - 75 years, median 41 years) with AML diagnosed and treated in West China Hospital from September 2009 to June 2010 were analyzed and followed up in the study. The leukemia-associated aberrant immunophenotype (LAIP) of all the patients were detected by multiparameter flow cytometry (FCM) before therapy. The level of MRD in the peripheral blood at day 8 of induction chemotherapy was detected by FCM based on the LAIP. The overall survival curve was drawn by calculation using Kaplan-Meier method using, and the comparison between different groups was carried out by Log-rank test. The results indicated that after first course therapy, the levels of peripheral D8RD in 7 out of 29 AML cases were lower than 0.01% (negative group), and that in another 22 cases were higher than 0.01% (0.08% - 55%, positive group). The sex, age, WBC, LDH, percentage of bone marrow blasts at diagnosis in these groups were not statistically different. 6 cases achieved CR (86%) in D8RD negative group, and also 6 cases achieved CR (27%) in D8RD positive group, CR rate in D8RD negative group was higher than in D8RD positive group (86% vs 27%, P < 0.05). The median follow-up of 29 cases lasted for 15 months; the 1-year overall survival rate of D8RD negative and D8RD positive groups was 100% and 39.4%, respectively (P < 0.01). It is concluded that MRD level in peripheral blood at day 8 of induction chemotherapy is an early index to predict clinical efficacy of induction therapy in AML.

Pre-Emptive Therapy with Modified Donor Lymphocyte Infusion Could Reduce Relapse and Improve Survival in Standard-Risk Acute Leukemia Patients After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 659Objects To evaluate the role of pre-emptive therapy with modified DLI on the prevention of relapse and improvement of survival after allo-HSCT. Methods The prospective study was performed to monitor MRD including WT1 and leukemia-associated aberrant immunophenotypes (LAIPs) in consecutive 814 standard-risk acute leukemia and MDS-RAEB patients after allo-HSCT, and then to initiate pre-emptive therapy with IL-2 (group B) or modified DLI (group C). Results The 3-year cumulative incidence of relapse, OS, DFS, and TRM were 18.1%, 66.0%, 61.6% and 19.7% for group A (without MRD); 68.0%, 23.9%, 20.8% and 11.2% for group B; as well as 29.8% (P=0.000), 55.4% (P=0.003), 52.5% (P=0.000), and 15.6% (P=0.208) for group C (Figure 1). In addition, the cumulative incidence of acute GVHD, grade 2 to 4 acute GVHD and grade 3 to 4 acute GVHD after MRD were 10.2%, 8.2% and 4.1% for group B; but 30.8% (P=0.017), 27.9% (P=0.017), and 8.4% (P=0.471) for group C. As well as, the 2-year cumulative incidence of chronic GVHD and chronic extensive GVHD after MRD were 37.3% and 30.5% for group B; as well as 42.9% (P=0.982) and 34.2% (P=0.858) for group C. Multivariate analysis identified pre-emptive therapy with modified DLI for the intervention of MRD (P=0.029, OR=2.016) as the only significant influence factor of superior survival. Furthermore, for the patients receiving modified DLI, multivariate analysis identified DLI-associated chronic GVHD (P=0.037, OR=6.158) and MRD conversion to negative within 3 months after DLI (P=0.042, OR=4.929) as the significant influence factors of lower relapse. Among the patients receiving modified DLI, the patients who had DLI-associated chronic GVHD and had MRD conversion to negative within 3 months after DLI, obtained the most superior survival (3-y relapse 0.0%, TRM 7.7%, OS 92.3%, and DFS 92.3%); however, the patients who had no DLI-associated chronic GVHD and had persistent MRD within 3 months after DLI, obtained the most inferior survival (3-y relapse 51.0% (P=0.045), TRM 20.4% (P=0.484), OS 28.6% (P=0.035), and DFS 27.4% (P=0.041)) (Figure 3). Moreover, apart from the patients who had MRD conversion to negative within 3 months after DLI and had DLI-associated chronic GVHD, repeated DLI possibly made other patients get better survival (P>0.05) (Figure 4). Conclusions This study for the first time confirmed that as compared with the use of IL-2, pre-emptive with modified DLI was more an effective method to prevent relapse and probably a superior method to improve DFS in standard-risk acute leukemia and MDS-RAEB patients after HSCT. Furthermore, for the patients without DLI-associated chronic GVHD, repeated DLI should be performed in order to induce chronic GVHD, so as to possibly make the patients get better survival. In addition, this study confirmed again that MRD monitoring with WT1 and LAIPs was a reliable measure to detect relapse of acute leukemia after HSCT, because of its high specificity and sensitivity. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Flow Cytometric Detection of Minimal Residual Disease In Acute Myeloid Leukemia – the Addition of hMICL and CD123 Add to Sensitivity and Applicability

AbstractAbstract 2719The outcome of acute myeloid leukemia (AML) is strongly correlated to the extent of minimal residual disease (MRD) at remission. Fusion transcripts, over-expressed genes and mutated genes have been employed in MRD quantification by RQ-PCR and are applicable for 30%–80% of all patients. More recently, flow cytometry (FCM) has been implemented as an additional tool for MRD detection employing strict gating criteria in detection of so-called leukemia associated aberrant immunophenotypes (LAIPs). In line with data from the Schuurhuis group we have identified human Myeloid Inhibitory C-type lectin (hMICL) as a candidate for AML marking at diagnosis and stable expression at relapse in a consecutive study of more than 150 patients. Importantly, we found expression of hMICL in the progenitor populations (defined as CD34+CD38- or CD117+CD38-) in AML cases. This contrasts to its absence on nearly all hematopoietic progenitors in healthy individuals and its lack of expression in acute lymphoblastic leukemia patients. Given that CD123 is also expressed in the vast majority of AML blasts at diagnosis and is characterized as a myeloid progenitor marker we hypothesized that the combination of hMICL and CD123 might serve as widely applicable MRD markers in AML and as such improve sensitivity for this approach. We performed a prospective study encompassing 38 patients with 74 follow up samples. Importantly, the assay was performed in strict parallel with other LAIPs, i.e. with no special provisions for the new markers. This FCM assay correlated strongly (88%) to available data from the most sensitive RQ-PCR marker of a series of molecular aberrations (p=0.37, McNemars chi^2 test), and that, the MRD detection and consequently reduced qualitative discordance resulted from a fourfold reduction of patients with FCM negative results but positive RQ-PCR result (table 1). The quantitative correlation coefficient between RQ-PCR and FCM increased from 0.46 to 0.7 by addition of hMICL and CD123 (figure 1).When sensitivities of the FCM assay were directly compared to those of validated RQ-PCR assays it was apparent that that of the FCM was inferior. However, it was still in the range in which it strongly predicted treatment failure. Undoubtedly, running more cells and samples and by employing more stringent gating and post-run analyses will further improve this strategy. However, given that treatment failures in AML are apparent by WT1 RQ-PCR with sensitivities at the 1; 1.000 range, the standard hMICL/CD123 assay should suffice in the vast majority of situations. We conclude that high-density expression of both hMICL and CD123 in the vast majority of AML cases at diagnosis and stable expression during the course of the disease combined with high sensitivity holds the potential for wide applicability and improved MRD monitoring by FCM using setup and interpretation not requiring special attention. This assay promises to be of great value in fast and efficient management of all AML patients. [Display omitted] Disclosure:No relevant conflicts of interest to declare.

Minimal Residual Disease Assessment by Flow Cytometry In AML Patients at Time of Aplasia Post-Induction Is Highly Predictive of Outcome and Strongly Correlates with Molecular MRD In a Subgroup of NPM1 Positive Patients

AbstractAbstract 1723 Background:Quantification of minimal residual disease (MRD) by multiparameter flowcytometry (MFC) in patients with acute myeloid leukemia (AML) provides significant prognostic information. Previous analyses have shown that positivity of leukemia-associated aberrant immunophenotype (LAIP) after induction and consolidation therapy is a risk factor concerning relapse and reflects over-all survival and relapse-free survival. Albeit molecular MRD monitoring has translated into therapeutic strategies in certain AML subgroups, e.g. APL, flowcytometric MRD assessment is still not integrated into therapeutic strategies. Methods:We performed a retrospective analysis of flowcytometric MRD data of 201 adult patients with AML. Patients were treated within the clinical study of the German AML cooperation group (AMLCG99 or sHAM); patients with APL were excluded. 165 patients were in the intermediate risk group according to karyotype; NPM1 and Flt3 positivity was present in 80 and 59 patients, respectively. 51% of patients relapsed in the course of disease, the median overall survival (OS) and relapse-free survival (RFS) were 518 and 292 days, respectively. MRD assessment by 3-color-flowcytometry was performed at initial diagnosis, during bone marrow aplasia (day 16 – 18 of induction therapy), after induction, after consolidation and at time of relapse. MRD flow was compared to molecular MRD assessment in patients with mutated NPM1. Results:Data on flowcytometric MRD assessment were available in 99% of patients at initial diagnosis, 74% on day16 of induction therapy, 80% post-induction, 42% post-consolidation and 62% at time of relapse. MRD positivity was defined as percentage of LAIP positive cells > 0,15%. In addition, the post-therapeutic degree of LAIP reduction compared to initial diagnosis was assessed: an indequate degree of LAIP reduction was set at a log difference (initial diagnosis/ day16) < 2. We could confirm that flowcytometric MRD predicts OS and RFS. The absolute level of MRD as well as the relative degree of MRD reduction on day16 of induction therapy defined significantly distinct risk groups concerning OS and RFS (OS: p = 0,03, RFS: p = 0,002). Discriminating the different types of LAIPs, we could demonstrate that cross-lineage phenotypes have the best sensitivity and specificity for predicting relapse (sensitivity 64%, specificity 88% on day16). The course of LAIP and NPM1 as assessed by rtPCR, highly correlates on day16 (correlation coefficient 0,84). Conclusion:Flowcytometric MRD assessment provides significant prognostic information at a very early point in induction therapy. In a subpopulation of NPM1+ AML patients we found a strong correleation of flowcytometric MRD and molecular MRD assessment. LAIP reduction on day 16 post-induction could therefore be integrated into prognostic models and can improve risk stratification especially in the absence of molecular MRD markers. Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease Status at Day +100 Post Allogeneic Hematopoietic Stem Cell Transplantation Is a Powerful Predictor for Post-Transplant Outcome In Patients with High Risk Acute Leukemia

AbstractAbstract 3550Background and Objectives Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still a major cause for the failure in treatment. It has been shown that there was a closely relationship between the level of minimal residual disease (MRD) and relapse in acute leukemia (AL) patients; However, the application of multiparameter flow cytometry (MFC) for MRD assessment in high risk patients with AL who undergoing allo-HSCT is little concerned. We retrospectively analysed the serial results of MRD of 52 high risk patients with AL to evaluates the prognostic value of MRD pre and post transplantation. Methods52 patients with a median age of 29 (13–55) years have been enrolled on this study in our hospital from January 2003 to September 2008.Diagnoses included AML (n=27) and ALL (n=25). The patients had been analyzed retrospectively the level of MRD pre-(day-30)and post-HSCT(day+30 and +100)using three color FCM with CD45/SSC gating and a comprehensive panel of monoclonal antibodies, at least one leukemia associated aberrant immunophenotype (LAIP) at diagnosis. According to the cutoff value 0.1%, two groups were defined based on the level of patient's MRD level< (low level group) or >= (high level group) 0.1%. ResultsThe median follow up were 23 (range 1–60) months. 1.MRD level declines significantly (P=0.03) post transplant. 2. There were significantly difference between low level and high level group at day -30 before transplant with 3 years event free survival(EFS) and relapse free survival (RFS)(77.4% and 88.4% vs. 22.3% and 25.7%, p=0.007and p=0.001 respectively). 3. Concerning about MRD at day +100 after transplant, outcome was significantly better among patients with low level MRD group versus high group including 3 years OS,EFS and RFS(84.2%, 79.5% and 89.5% versus 22.9%, 9.5% and 11.2%).4. The median time from high level MRD detected first time to clinical relapse was 2.5 (range from 1 to 33) months in relapsed patients. 5. The patients with cGVHD had better 3 years OS and EFS than that without cGVHD(86.3% vs 12.1%, p<0.001 and 65.3% vs.14.8%, p< 0.001 respectively). 6. Multivariate Cox regression analysis revealed that MRD on day +100 as well as chronic GVHD were independent parameters predictive for OS and EFS. ConclusionsMRD monitoring pre- and post-transplant is an important tool to predict the outcome of transplantation for patients with high risk AL. The MRD check point at day +100 should be considered crucial for subsequent therapeutic decisions after allogeneic transplantation. Disclosures:No relevant conflicts of interest to declare.

Prognosticating of Relapse Risk Based on Multiparameter Flow Cytomeric Assessment of Minimal Residual Disease in Patients with Acute Myeloid Leukemia

Abstract Objective: To investigate the prognosticating of relapse risk based on assessment of minimal residual disease(MRD) in patients with acute myeloid leukemia(AML).Methods: Multiparameter flow cytometry (MPFC) analysis were used to detect the leukemia-associated aberrant immunophenotype (LAIP)of the pretreated patients with AML and assess the level of MRD after remission induction(Post-Ind MRD) and consolidation therapy(Post-Cons MRD).Results: The results showed that definite LAIP could be detected in about 94.3%of patients (115/122) with AML (except APL). Only one LAIP was identified in 15 cases (13.0%), but in other 100 cases (87.0%), two or more LAIP were identified. The most frequent LAIP identified was cross-lineage antigen expression (40.9%). The percentages of asynchronous antigen expression, antigen over-expression and antigen lack expression were 20.9%, 27.0% and 34.8% respectively. MRD frequency was monitored in 41 CR patients with AML after remission induction chemotherapy and 2 or more cycles of consolidation chemotherapy. 24 patients were Post-Ind MRD+ and 17 patients were Post-Ind MRD−. The percentages of relapse in Post- Ind MRD+ and Post-Ind MRD− were 75.0%(18/24) and 29.4%(5/17) respectively after consolidation chemotherapy. The relapsed free survival(RFS) of the patients with Post-Ind MRD+ and Post-Ind MRD− was 49.06±6.53 months and 11.92±1.64 months(P<0.0001) respectively. 18 patients were Post-Cons MRD+ and 23 patients were Post-Cons MRD−. The percentages of relapse in Post-Cons MRD+ and Post-Cons MRD− patients were 100%(18/18) and 21.7%(5/23) respectively after consolidation chemotherapy. The RFS of the patients with Post-Cons MRD+ and Post-Cons MRD− was 41.74±5.52 months and 10.06±1.72 months (P<0.0001) respectively.Conclusions: The level of post-Ind MRD and post-Cons MRD identified in the patients with AML was highly associated with their RFS. MRD measured by MPFC provided prognostic information in AML patients.

CD11b Is a Therapy-Induced Marker for Monitoring Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia.

Assessment of minimal residual disease (MRD) has become central to the clinical management of patients with acute lymphoblastic leukemia (ALL). Among the methods available for MRD monitoring, flow cytometry (FCM), which relies on the presence of leukemia-associated aberrant immunophenotype, holds great promise for clinical application. However, the major antigens used for FCM-MRD identification (CD10, CD34, CD45, CD20, CD19, TdT) undergo expression changes during therapy. Moreover, the presence of normal hematopoietic progenitors, in particular in the regenerating bone marrow after treatment, negatively impacts the sensitivity and specificity of MRD detection. Recently, we analysed genome-wide gene expression in blasts isolated from peripheral blood of pediatric precursor B-cell (PBC)-ALL patients after one week of therapy (day 8 cells). Expression changes observed in the day 8 cells pointed to several cell surface molecules, whose expression has not been characteristic for B-lineage hematopoiesis. In particular CD11b surface antigen has been frequently up-regulated in the day 8 cells. In the present study, we addressed expression dynamics of CD11b in PBC-ALL at clinically significant MRD timepoints during induction therapy (days 15, 33 and 78; ALL-BFM protocol). To this end, a CD11b specific antibody has been included into a nine-color, single-tube panel (antibodies to CD19, CD20, CD10, CD34, CD45, CD58, CD3, and a nuclear stain Syto16), which has been applied in order to detect residual blasts among 106 cells in bone marrow specimens from patients with PBC-ALL. At day 15, mean expression of CD11b (in MESF units) has been significantly increased if compared with leukemic cells at diagnosis (9600+/−2800 vs 850+/−140; p=0.005). The up-regulation by more than 10-fold has been found in 8 of 24 cases (33%), and has reached, in part, very high levels (eg, 450 MESF vs 49500 MESF at diagnosis and day 15, respectively). This indicates that CD11b expression changes are due to a therapy-induced gene up-regulation rather than to a clonal selection during clinical treatment. At the later timepoints of induction therapy, 7 of 22 patients (day 33) and 2 of 18 patients (day 78) were MRD positive. CD11b expression, if increased at day 15, retained its high values on day 33 (7900+/− 3200 MESF, 6 patients) and on day 78 (15100 MESF, 1 patient). Importantly, in contrast to leukemic cells, their normal CD19+CD10+ counterparts in both, non-leukemic and ALL bone marrow samples, remained CD11b negative. This difference has facilitated a reliable discrimination of normal and leukemic blasts in the MRD positive cases with regenerating bone marrow at day 78. In conclusion, treatment-induced up-regulation of CD11b in PBC-ALL has a promising potential as a novel marker, which may considerably improve specificity of FCM-MRD detection in bone marrow samples with a complex hematopoietic background.

Use of five-color staining improves the sensitivity of multiparameter flow cytomeric assessment of minimal residual disease in patients with acute myeloid leukemia

Application of five-color staining may improve quantification of minimal residual disease by multiparameter flow cytometry in acute myeloid leukemia. We analysed bone marrow samples in 139 cases using a comprehensive antibody panel with five-color combinations. Sensitivity was estimated by quantification of leukemia-associated aberrant immunophenotype (LAIP)-positive cells for each LAIP in 18 normal bone marrow (BM) samples. The logarithmic difference (LD) in LAIP-positive cells between leukemic and normal BM amounted to a median of 3.32 (range 1.76 – 4.89). Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells while percentages of LAIP-positive leukemic cells changed only marginally (median gain in LD = 0.54; maximum gain = 3.30). Because regenerating bone marrow has not been used as control data are most important to post-therapy checkpoints. In 32 patients with clinical follow-up, a LD higher than the median (3.25) at the follow-up checkpoint corresponded to a longer event-free survival. These data suggest that the application of five-color staining significantly improves the sensitivity and accuracy of the method.

Multivariate Analysis of Prognostic Impact of Flow Cytometrically Determined Minimal Residual Disease in Patients with Acute Myeloid Leukemia.

Treatment of acute myeloid leukemia (AML) is adapted according to individual risk profiles. The assessment of levels of minimal residual disease (MRD) is feasible in the vast majority of patients with AML and may significantly improve prognostication. We applied multiparameter flow cytometry to highly sensitively quantify MRD in patients with AML and analyzed its prognostic impact in a multivariate analysis. A total of 356 patients receiving standardized intensive antileukemic treatment was analyzed at different checkpoints: CP1 (up to day 28, n=156), CP2 (day 28–60, n=122), CP3 (day 61–120, n=195), CP4 (day 121–365, n=172), and CP5 (after day 365, n=73). Leukemic cells were identified by their individually defined leukemia-associated aberrant immunophenotypes (LAIPs). MRD levels were calculated as the logarithmic difference (LD) between LAIP-positive cells at diagnosis and LAIP-positive cells at follow-up assessment. The median LD amounted to 2.18 (range, −0.14 to 4.20) at CP1, 2.31 (−0.03 to 4.17) at CP2, 2.49 (0.11 to 4.17) at CP3, 2.58 (−0.28 to 4.28) at CP4, and 2.87 (0.46 to 4.02) at CP5. A higher LD (continuous variable) was related to a better event-free survival (EFS; CP1, p=0.0002; CP2, p=0.00001; CP3, p=0.0002; CP4, p<0.00001; CP5, p=0.00007) and to a better overall survival (OS; CP1, p=0.004; CP2, p=0.001; CP3, p=0.021; CP4, p=0.00006). Other parameters related to EFS and OS were age and cytogenetics in the present series. The prognostic impact of MRD levels on outcome was idependent of cytogenetics and age for EFS (CP2 to CP5) and OS (CP2 and CP4). MRD was the most important prognostic parameter at CP4 and CP5. Separation of patients into two groups, respectively, by the median LD resulted in significant differences in EFS at all CPs and in OS at CP1 to CP4. The largest difference was observed at CP4: median EFS, 57.1 vs. 13.7 months, p<0.00001; 3-year-OS, 95% vs. 65%, p=0.0003. Determination of MRD levels by multiparameter flow cytometry provides a highly powerful and independent prognostic parameter which is applicable to the total of an AML population. It should be evaluated as a stratification parameter in clinical trials.