Amount Of Minimal Residual Disease Research Articles

The Pathogenesis of Multiple Myeloma (MM) Is Preceded By Mutated Lymphopoiesis and B Cell Oligoclonality That Persist in Patients with Negative Minimal Residual Disease (MRD)

The Pathogenesis of Multiple Myeloma (MM) Is Preceded By Mutated Lymphopoiesis and B Cell Oligoclonality That Persist in Patients with Negative Minimal Residual Disease (MRD)

PF178 DEFINITION OF REPRESENTATIVE BONE MARROW SAMPLE BASED ON PARALLEL EVALUATION OF PERIPHERAL BLOOD DURING THE THERAPY OF ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Acute lymphoblastic leukemia (ALL) treatment leads to elimination of the blasts and subsequent regeneration of non‐malignant populations. Amount of minimal residual disease (MRD) is the strongest prognostic marker and its value differs between bone marrow (BM) and peripheral blood (PB). Therefore for patients stratification is necessary to evaluate non‐hemodiluted BM sample. Our AIEOP BFM 2009 protocol considers a BM sample non‐hemodiluted if >2% of its cells are erythroid precursors (EP). EPs are defined as CD19neg(orCD7neg)CD45neg cellsAims:1. To evaluate utility of the currently used marker of BM representativeness.2. To define new, more exact approach for definition of representative BM.Methods:Patients (n = 528) diagnosed with ALL between 2007 and 2015 were included into the study. We analyzed proportion of EPs defined as CD19neg(orCD7neg)CD45neg in BM and in parallel also in PB at day 15 together with viability dye DAPI. We tested whether a new definition of EPs including an added CD71 marker (transferrin receptor expressed since early phases of erythropoiesis) improves its specificity.Then we evaluated other non‐malignant populations (n = 342) in BM and PB for the new definition of representative BM (lymphocytes, granulocytes, monocytes, erythroid precursors, mature B cells, T cells and NK cells in both B cell precursor and T ALL; CD4+, CD8+ and CD4negCD8neg T lymphocytes in T ALL only).Results:1. We identified high proportion of apoptotic cells in EPs defined as CD19neg(orCD7neg)CD45neg (6.5–96%, median 55%) The population defined as CD19neg(orCD7neg)CD45negCD71pos was more viable (apoptotic cells 0–66%, median 9%) p < 0.0001. Then we evaluated population CD19neg(orCD7neg)CD45neg in PB. We found that as many as 29% of PB samples have >2% of EP (the value that is currently used as a marker of BM without hemodilution).2. We defined a new assessment of BM representativeness based on comparison of non‐malignant populations between BM and PB. With increasing difference between the proportion of any population between BM and PB, the possibility of hemodilution lowers. According to this approach we can calculate maximal possible hemodilution and its influence on stratification of the patient. We evaluated 342 patients with available paired BM‐PB samples and identified 6 patients with highly hemodiluted BM samples who might have been misstratified.Summary/Conclusion:We defined a new, more accurate approach for assessment of BM representativeness by comparison of non‐malignant populations between PB and BM. By this approach we can exclude hemodilution in majority of the samples.The currently used marker of representativeness – proportion of EPs, defined as CD19neg(orCD7neg)CD45neg – is not sufficient for assessment of BM representativeness. If proportion of EPs is required, CD71 should be added for the definition.Supported by grants NV18–03–00343, NV18–07–00430 and LO1604.

Prediction of Relapse By microRNA Expression in Pediatric B-Lineage Acute Lymphoblastic Leukemia

Aim: microRNAs (miRs) have been implicated in many malignancies. Our aim was to identify specific miRNAs that can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated on BFM protocols, better than the current risk group stratification. Currently, the main method for risk group stratification is based on the amount of minimal residual disease (MRD) assessed at specific time points by real time quantitative PCR (RQ-PCR).Material and methods: Following microRNA expression analysis performed on 48 bone marrow samples at diagnosis, we focused on several miRs that correlated with at least 3 of the established prognostic markers in ALL to be validated in a cohort of 138 B-lineage ALL samples. Of the miRs studied, down regulated miR-151-5p and miR-451 and upregulated miR-1290 significantly correlated with outcome.Results: Low expression of miR-151-5p, miR-451 or of both together resulted in significantly worse relapse free survival (RFS) (56%, 63% and 47%, respectively) compared to RFS rates when either or both miRs were highly expressed (80%, 78% and 78%, respectively) (p=0.007, 0.042 and 0.002, respectively). High expression of miR-1290 resulted in worse RFS compared to those that expressed low levels (54% versus 81%; p=0.014). When combining the 3 miRs, a patient expressing low levels of both miRs and/or high levels of miR-1290 had a RFS of 54% (p=0.004). Furthermore, the expression of the 3 miRs could distinguish within the TEL-AML1 negative cohort two groups; one with 80% and the other with 44% RFS (p=0.004). Multivariate Cox regression analysis identified low expression of both miRs and/or high expression of miR-1290 as an independent prognostic marker in the PCR-MRD non-high risk cohort. Patients expressing abnormal levels of the 3 miRs had a 4.47-fold increased risk for relapse (p=0.037). Deletion of IKZF1 gene is a known adverse prognostic marker in B-lineage ALL. In 85 patients, IKZF1 status was analyzed and 9 patients were found to harbor the deletion. The expression of the 3 miRs could still identify an adverse group of patients with 69% RFS within the group with no deletion (p=0.004). When adding IKZF1 and TEL-AML1 status to the Multivariate analysis in addition to PCR-MRD, the expression of the 3 miRs remained a significant independent marker with a 24 fold increased risk for relapse (p=0.011). [Display omitted] Conclusion: Our results identify the combination of miR-151-5p, miR-451 and miR-1290 as a novel biomarker for outcome in pediatric B-lineage ALL patients, already at time of diagnosis. This may lead to improved risk group stratification and enable early therapeutic intervention that may result in better RFS. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic but Not Autologous Stem Cell Transplant Attenuates the Negative Prognostic Impact Dictated By Pretransplant MRD Positivity

Multiparametric flow cytometry (MPFC) detection of minimal residual disease (MRD) represents a robust surrogate for the quality of complete remission (CR) and reliably predicts clinical outcome. In our experience, MRD detection provides prognostically relevant information when assessed at the post-consolidation time point. Ten years ago we demonstrated that the amount of MRD before autologous stem cell transplant (AuSCT) affected outcome. More recently, other authors have extended this observation to allogeneic stem cell transplant (ASCT) showing that pre-transplant MRD is a major determinant of prognosis regardless of graft-versus-leukemia (GVL) effect. The aim of our study was to evaluate, in an extended series of patients submitted to AuSCT or ASCT, the impact of a pre-transplantation MRD positive (MRDpos) or negative (MRDneg) status on overall survival (OS) and disease free survival (DFS). We analyzed 173 MRDpos and 53 MRDneg patients of whom 67 were submitted to AuSCT and 51 to ASCT. Eighty-two patients received no transplant because of age, poor performance status or insufficient stem cell harvest whereas 26, all in the MRDpos group, relapsed before transplant delivery. In the AuSCT group, before transplant, 32/67 (48%) were MRDneg and 35/67 (52%) MRDpos, with MRDneg group showing a superior OS (55% vs 20%, p=0.007). In the ASCT group, before transplant, 45/51 (88%) were MRDpos and 6/51 (12%) MRDneg. For 21 out of 51 (41%) sources of stem cells were matched unrelated donors (12) or haploidentical donors (9). In this subgroup, MRDpos and MRDneg patients shared a comparable 5-years OS (60% vs 56%, p=NS), with a 36% survival gain for those MRDpos who received ASCT as compared to AuSCT. Among MRDneg patients, no survival differences were demonstrated between those submitted to AuSCT or ASCT (55% vs 60%, p=NS). Such a lack of difference is likely due to the higher treatment related mortality (ASCT 3/6, 50% vs AuSCT 2/32, 6%, p=0.003) which counterbalanced the lower relapse rate in the ASCT group (ASCT 0/6, 0% vs AuSCT 11/32, 34%, p=NS). In conclusion, ASCT confers a significant survival advantage to MRDpos patients, attenuating the negative prognostic impact of pre-transplant MRD positivity. ASCT may expose MRDneg patients to an excess of toxicity suggesting that in these patients the allogeneic option should be postponed after a second remission. In MRDpos patients, AuSCT does not represent a valid therapeutic choice and ASCT, which should be timely delivered, also considering alternative sources of stem cells. DisclosuresNo relevant conflicts of interest to declare.

Abstract B34: Increased risk of relapse in non-high-risk children with pediatric B-lineage acute lymphoblastic leukemia can be predicted at diagnosis by microRNA expression

Abstract Aim: microRNAs (miRNAs) have been implicated in many malignancies. Our aim was to identify specific miRNAs that can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated on BFM protocols, better than the current risk group stratification. Currently, the main method for risk group stratification is based on the amount of minimal residual disease (MRD) assessed at specific time points by real time quantitative PCR (RQ-PCR). Material and methods: Following microRNA expression analysis, we decided to focus on miR-151-5p and miR-451 that significantly correlated with known prognostic factors in ALL. Validation was performed by measuring the expression levels of miR-151-5p and miR-451 by RQ- PCR on a cohort of B-lineage ALL patients treated by a Berlin-Frankfurt-Munster (BFM) protocol following exclusion of Philadelphia positive patients (n=123). Results: Low expression of miR-151-5p, miR-451 or of both together resulted in significantly worse relapse free survival (RFS) (61%, 64% and 43%, respectively) compared to RFS rates when either or both miRNAs were highly expressed (83%, 82% and 82%, respectively) (p=0.023, 0.03 and 0.0001, respectively). Multivariate Cox regression analysis identified low expression of both miRNAs as an independent prognostic marker. Patients expressing low expression levels of both miRs had a 8.8-fold increased risk for relapse (p=0.003). Deletion of IKZF1 gene is a known adverse prognostic marker in B-lineage ALL. In 88 patients, IKZF1 status was analyzed and 9 patients were found to harbor the deletion. The expression of both miRs could still identify an adverse group of patients with only 50% RFS within the group with no deletion (p<0.0003). Even in a non-BFM treated B-lineage ALL cohort from The Netherlands, the expression levels of both miRs significantly correlated with outcome (p=0.003). Conclusion: Our results identify miR-151-5p and miR-451 as novel biomarkers for outcome in pediatric B-lineage ALL patients, regardless of treatment protocol. This may lead to improved risk group stratification and better RFS. Citation Format: Smadar Avigad, Iedan RN Verly, Gertjan JL Kaspers, Jacqueline Cloos, Anat Ohali, Michal Hameiri-Grossman, Keren Shichrur, Eva Fronkova, Jan Trka, Drorit Luria, Yona Kodman, Hadar Mirsky, Dafna Gaash, Marta Jeison, Galia Avrahami, Sarah Elitzur, Gil Gilad, Batia Stark, Isaac Yaniv. Increased risk of relapse in non-high-risk children with pediatric B-lineage acute lymphoblastic leukemia can be predicted at diagnosis by microRNA expression. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B34.

Assessment Of Minimal Residual Disease In Acute Myeloblastic Leukemia In Multiparameter Flow Cytometry

Acute myeloid leukemias (AML) represent a vast and complex group of diseases where numerous molecular lesions have been and keep being described. From the immunophenotypic point of view, probably because of the variety of cells in the myeloid lineage, a rather large variety also exists. The detection of minimal residual disease (MRD) in such conditions therefore meets the challenge of tracking the proper anomaly and correctly separate leukemic cells from their normal counterparts. In an oligocentric project initiated in France in late 2006, ten cytometry platforms and six molecular biology laboratories collaborated to detect MRD concomitantly in flow and with molecular techniques. The flow cytometry part of this work is reported here.A total of 307 consecutive patients were tested at diagnosis with a comprehensive common panel allowing for the detection of immature markers and potential leukemia-associated immunophenotypic patterns. Follow-up samples were planned to be obtained after induction and at the end of treatment, with an optional control before the second consolidation. In fine, 274 patients had at least one point of follow up. All samples were tested in a technique of whole bone marrow lysis no wash, avoiding any cell loss. The flow cytometry panel comprised ten five-colors tubes, all containing CD45 as gating marker. A newly developed strategy was devised to analyse MRD data. The approach of the GTLLF (Arnoulet, Cytometry part B, 2011) was first applied to properly identify mature polymorphonuclears, monocytes and lymphocytes, allowing for a negative Boolean selection of immature cells in the region dubbed “bermudes” by this group. Focusing on this area, each combination of the four markers tested together with CD45 was then displayed in a total of six biparametric histograms. For each of them, on the diagnosis sample, quadrant gates were constructed so that the lower left one contained no blast cells. A Boolean operation was then designed to exclude all these six areas, thereby combining the positive blasts present in the three other parts of each quadrant. The resulting population was visualized on a CD45/side scatter biparametric histogram to check that the cells appeared as a focused cluster at a precise position. The same strategy was then applied for each patient's consecutive samples, always checking whether any cells identified with this protocol displayed the scattered pattern of cells engaged in maturation (no MRD) or constituted a focused population without maturation (positive MRD). The amount of MRD was then calculated taking into account as denominator the whole population of nucleated cells in the sample (excluding debris on a live gate). As internal control a specific feature of the Kaluza software was used to merge samples obtained for a given patient in order to display on the same worksheet the diagnosis and follow up samples using the principal component separation provided by the “radar” tool of this software. This original method proved to be easily applicable and provide a consistent help for MRD interpretation.All patients could be assessed for MRD with only two of the ten tubes used. These contained the following combinations : CD15, CD13, CD33, CD34, CD45 and CD7, CD117, CD33, CD34, Cd45. At diagnosis, any combination of expression of CD13, CD33, CD117 and CD34 could be observed, the percentage of positive cases for each of these antigens being respectively 86%, 89%, 81% and 58%. As a whole, 93% of the follow-up samples (MRD) tested contained less than 5% of cells with an immunophenotype comparable to that of diagnosis. This figure was 77% for less than 1% and 43% for less than 0.1%. The strategy devised for files analysis was easily applicable for all patients except those with myelomonocytic leukemia. For some of them, separation of the blasts from the monocytic compartment could be problematic in regenerating bone marrow samples.In conclusion, the flow cytometry part of this multicenter study allowed to establish that the combination of CD45 with CD13, CD33, CD117 and CD34 with the additional information provided by CD5 and CD7 represents a quasi-universal panel, now easy to implement on instruments with 8 or 10 detectors, for the detection of MRD in multiparameter in flow cytometry. Moreover, a powerful strategy of listmodes analysis was developed allowing for the direct comparison of several samples from the same patients and/or of a given sample and normal (control) bone marrow. Disclosures:No relevant conflicts of interest to declare.

Low Levels of Mir-151-5p and Mir-451 Predict Relapse in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

AbstractAbstract 2507 Aim:microRNAs (miRNAs) have been implicated in many malignancies. Our aim was to identify specific miRNAs that can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated according to BFM protocols already at diagnosis. The current risk group stratification is based on the amount of minimal residual disease (MRD) assessed at specific time points by real time quantitative PCR (RQ-PCR). Material and methods:Following miRNA expression analysis, we decided to focus on miR-151-5p and miR-451 that significantly correlated with known prognostic factors in ALL. Validation was performed by measuring the expression levels of miR-151-5p and miR-451 by RQ-PCR on bone marrow samples at diagnosis of 101 B-lineage ALL patients excluding Philadelphia positive patients. Results:Low expression of miR-151-5p, miR-451 or of both together resulted in significantly worse relapse free survival (RFS) (43%, 58% and 38%, respectively) compared to RFS rates when either or both miRNAs were highly expressed (81%, 75% and 75%, respectively) (p=0.001, 0.044 and 0.001, respectively). Moreover, following PCR-MRD stratification, low expression of miR-451 or both miRNAs remained significant within the PCR-MRD medium risk group (p=0.00001) and within the standard group for both miRNAs (p=0.024). Multivariate Cox regression analysis identified low expression of both miRNAs as an independent prognostic marker with 11.7 fold increased risk for relapse (p=0.002). After excluding patients harboring the adverse genetic markers Ikaros deletion and P2RY8-CRLF2 rearrangement, a patient expressing low levels of both miRs had a 35 fold risk to relapse (p=0.005).Analyzing a non-BFM treated B-lineage ALL cohort from The Netherlands, both miRNAs significantly correlated with outcome (p=0.003). Conclusion:Our results identify miR-151-5p and miR-451 as novel biomarkers for outcome in pediatric B-lineage ALL patients, regardless of treatment protocol. This may allow earlier and improved risk group stratification already at diagnosis, enabling exploration of early therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

The utility of multiparametric flow cytometry for the detection of minimal residual disease in acute lymphoblastic leukemia

The quantification of the amount of minimal residual disease (MRD) in the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL) at several points of the treatment of the disease has proven to be an important independent predictor of treatment outcome(1-5). More recently, treatment protocols have been designed to adjust chemotherapy according to the presence or absence of MRD on day 8 or 15 of the induction therapy and at the end of induction (day 29) in order to deliver sufficient chemotherapy to cure the patient and minimize acute and long-term side effects. However, this approach is only justified in controlled clinical studies. Flow cytometry, an essential component of the diagnostic work-up in ALL, is used to determine the cell lineage of leukemic blasts, to quantify antigen expression and to detect aberrant antigen expressions. These data are essential for the detection of abnormal cell populations in the BM in the study of MRD. It has been shown that leukemic blasts may be distinguished from normal lymphoid precursors in up to 87% of ALL cases. Moreover, if the technique is well standardized, it is able to detect one abnormal cell among 10,000 to 100,000 normal BM cells(3). Several different panels of antibody combinations have been described with some also considering the cost-effectiveness and simplicity(1,5). However, combinations of at least four colors are essential to give reliable results. The following figure presents the analysis of a case of B-cell ALL. The abnormal blasts may be distinguished from the normal ones by their deficient and variable expression of CD45 (Figure 1). Figure 1 Minimal residual disease study of a B-cell acute lymphoblastic leukemia case. Phenotype at diagnosis: CD45dim variable, CD19+, CD10+ and aberrant expression of CD13/ CD33. Residual blasts in yellow (dim and variable expression of CD45, positive for CD19, CD10 ...

3.2 Evolution of High-Sensitivity, Multi-Color Flow Cytometric Immunophenotyping for Minimal Residual Disease Detection in Chronic Lymphocytic Leukemia: Peripheral Blood versus Bone Marrow?

Background: Detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) patients has become increasingly important as modern chemoimmunotherapy (CIT) regimens have become effective in reducing tumor burden or even eliminating detectable disease. From a clinical perspective, MRD status posttreatment has been shown to predict durability of remission. Current 6and 8-color flow cytometry assays can easily detect CLL cells to the 0.005% level (1 in 20,000 events). Historically, peripheral blood (PB) has been the standard source for evaluating CLL, while bone marrow (BM) studies have been used primarily to evaluate patients with unexplained cytopenias. A previous study based on 4-color flow cytometry analysis suggested a high concordance (87%) of MRD analysis between PB and BM as long as patients had not received monoclonal antibody therapy in the preceding 8-12 weeks. The goal of the present study was to compare MRD detection in PB and BM from CLL patients after first-line CIT using a highly sensitive 6-color flow assay to determine whether there were any differences in MRD levels. Methods: Thirty-one paired PB and BM specimens were obtained from 25 patients with a confirmed diagnosis of CLL. Twenty-one paired samples were obtained from 15 previously untreated patients on a phase II clinical trial evaluating lenalidomidebased consolidation therapy after 6 cycles of pentostatin, cyclophosphamide, and rituximab induction therapy. Ten paired samples were from previously untreated patients on a phase II clinical trial evaluating pentostatin, cyclophosphamide, and ofatumumab. Twentyeight paired specimens were obtained 3 months after rituximab/ ofatumumab and the remaining 3 specimens were obtained 8-12 weeks following rituximab therapy. Individual patient samples were processed and analyzed on the same day. High-sensitivity flow cytometric immunophenotyping was performed in all specimens. A total of 500,000 events were collected in all cases and analyzed on a Becton-Dickinson Canto II flow cytometer. The standard single-tube, 6-color antibody panel included CD45, CD19, CD20, CD5, and kappa and lambda immunoglobulin light chains; additional antibodies were added as needed. Positive events were based on the identification of lymphoid cells by light scatter followed by gating on CD19positive B-cells, then dual expression of CD20(lo)/CD5 cells, and finally evaluation for kappa/lambda monoclonality. The level of MRD was assessed by percentage MRD calculated as (CLL events)/ (total live, non-aggregated WBCs) 100. This assay was developed in a clinical laboratory and validated by comparing it to a previous standard 4-color assay in 562 specimens (unpublished data). Validation studies confirmed a higher level of sensitivity with the 6-color assay and the ability to consistently detect MRD or monoclonal B-cell lymphocytosis events to the 0.005% level (25/500,000 events). Results: PB and BM MRD analysis was concordant in 20/31 (65%) paired samples using the single-tube, 6-color antibody analysis; 15/31 paired samples had detectable MRD in both PB and BM, and 5 had no detectable MRD in either PB or BM. Eleven of 31 (35%) studies showed detectable MRD in BM, but no CLL cells in PB. Considering BM MRD analysis as the standard, the sensitivity of the PB MRD assay in this cohort was 58% (15/26). The amount of MRD was on average 5-fold greater in BM as compared to PB. The level of MRD in BM (range 0.006-6.008%; median 0.765%) was greater than in PB (range 0.035-38.726%; median 0.161%) in all cases, with a median difference of 0.343% (p 0.0054). In the 15 paired specimens with MRD in both PB and BM, the level of residual disease was 1% in 12/15 PB and 10/15 BM specimens. Among the 11 paired samples in which MRD was only detected in BM, 10 (91%) had 1% clonal B-cells present on the BM MRD assay, with positivity ranging from 0.005% to 3.231% (median 0.061%); this was 10 times lower than the median value of BM MRD for samples in which both PB and BM were MRD-positive, indicating a lower level of disease burden in the group with discordant MRD results on BM and PB analysis. Conclusion: A single-tube, 6-color flow assay is a highly sensitive approach for the detection of MRD in CLL. The level of MRD following effective CIT was 5-fold higher in BM as compared to PB. A negative PB MRD study cannot be used as a surrogate for the absence of BM disease in CLL patients.

Bone marrow aspiration technique may have an impact on therapy stratification in children with acute lymphoblastic leukaemia

Morphological evaluation of early response to chemotherapy and measurement of minimal residual disease by flow cytometry or PCR are being used for evaluation of prognosis and treatment stratification in children with acute lymphoblastic leukaemia (ALL). In a series of 14 consecutive bone marrow investigations from children with precursor B-cell ALL, morphological evaluations of smears and flow cytometric measurements of minimal residual disease in sequentially aspirated small (2 ml) and large (5-10 ml) volumes of bone marrow were compared, at various time points during therapy. The density of nucleated cells was markedly reduced in the large volume aspirate. The percentage of erythroblasts measured by flow cytometry was smaller, indicating dilution with peripheral cells. Similarly, the blast percentage was reduced with 54% in large aspirates, and in four instances with minimal residual disease of >0.1% in the small volume, the level of blasts in the large aspirate was below this limit. The amount of minimal residual disease should be measured in the first 2.5 ml of bone marrow aspirated from one puncture site. The procedure should be performed by experienced and carefully instructed doctors. In large aspirates, minimal residual disease will be underestimated, which may lead to failure to undertake a required intensification of therapy and a lower fraction of high risk patients in the trial.

Prognostic Significance of the Lost of a Major Molecular Response In Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia (Ph+CML).

AbstractAbstract 3435 Background:Albeit of well-known, dramatic improvements, there remain some questions to be solved around Ph+CML in treatment with tyrosine kinase inhibitors (TKI). Among these, the significance of the amount of minimal residual disease (MRD) measured by RT-PCR. For instance, loss of a so-called major molecular response (MMR) is claimed to be a Òsuboptimal responseÓ and following the ELN recommendations, a change in treatment should be considered in these patients. Aims:To evaluate the relevance of a loss of MMR in patients with complete cytogenetic response (CCR). Study Group and Methods:We have analized 81 patients treated with imatinib for CML in chronic phase with a median follow up of 66 months. 36 patients started imatinib after interferon failure and 45 as front line therapy. Major Molecular Response (MMR; BCR-ABL/ABL ratio<0.1% IS) at any time was achieved by 63 patients. Results:22 patients (34%) lost MMR (documented al least twice). The risk of losing MMR was higher in late MMR (>18 months) compared with those cases whose MMR came much earlier (<18 months): 70% vs 18% (p=. 000). We have found no correlation among the lost of MMR and classical prognostic factors (Sokal-Index, mutations at the TK domain or imatinib plasma levels). Of these 22 patients, 7 (32 %) recovered MMR later with no therapy changes, 8 (36%) experienced fluctuations in the BCR-ABL transcript-levels without losing CCR, 4 (19%) did not attain a MMR but remained in stable CRR, and 3 (13%) lost CCR. These regained MMR after being treated on second generation TKI. The results show how the stability of the early MMR is greater than late MMR (table1). Conclusions:In our experience, one third of the patients who lost MMR recovered it later on the same treatment. And only 13% went on to treatment failure. Perhaps some similar cases (after first losing MMR) should be closely monitored before a change in treatment. Also of note is, of course regarding only our experience, that the risk of a loss of MMR seems to be maximal in patients who achieve a late MMR.Table 1Evolution of patients after lossing MMREarly MMR (n= 9)Late MMR (n=13)Recover MMR45% (4)23% (3)Fluctuations33% (3)38% (5)Remained in Suboptimal Response11% (1)23% (3)Lost CCR11% (1)15% (2) Disclosures:No relevant conflicts of interest to declare.

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

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.

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ ABL x 10(4) also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with < or = 0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels < or = 10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia

Molecular monitoring by quantitative PCR techniques of residual leukaemia cells during the first phases of treatment can predict outcome in children with acute lymphoblastic leukaemia. We did a retrospective study of 30 children who had been treated according to the ALL-REZ BFM trials to assess whether amount of minimal residual disease during the first stages of treatment for relapsed acute lymphoblastic leukaemia could predict outcome. In children with minimal residual disease of less than 10(-3) at day 36, probability of event-free survival was 0.86 (95% CI 0.77-0.95), compared with 0 in children with minimal residual disease of 10(-3) or greater (p<0.001). Our results suggest that information about molecular response to treatment can be used to predict long-term outcome in relapsed childhood acute lymphoblastic leukaemia.

Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood

Sensitive techniques for detection of minimal residual disease (MRD) at degrees of one leukaemic cell per 10(3)-10(6) cells (10(-3)-10(-6)) during follow-up of children with acute lymphoblastic leukaemia (ALL) can provide insight into the effectiveness of cytotoxic treatment. However, it is not yet clear how information on MRD can be applied to treatment protocols. We monitored 240 patients with childhood ALL who were treated according to national protocols of the International BFM Study Group. 60 patients relapsed and the patients in continuous complete remission (CCR) had a median event-free follow-up of 48 months. Bone-marrow samples were collected at up to nine time points during and after treatment. Standardised PCR analysis of patient-specific immunoglobulin and T-cell receptor gene rearrangements and TAL1 deletions were used as targets for semiquantitative estimation of MRD. Amount of MRD was classed as 10(-2) or more, 10(-3), and 10(-4) or less. MRD negativity at the various follow-up times was associated with low relapse rates (3-15% at 3 years), but five-fold to ten-fold higher relapse rates (39-86% at 3 years) were found in MRD-positive patients. The distinct degrees of MRD appeared to have independent prognostic value (p [trend]<0.001) at all separate time points, especially at the first two time points (at the end of induction treatment and before consolidation treatment). At these two time points a high degree of MRD (> or = 10(-2)) was associated with a three-fold higher relapse rate when compared with patients with a low degree of MRD (< or = 10(-4)). At later time points (including the end of treatment) even a low degree of MRD was associated with a poor outcome. Positivity in patients in CCR after treatment was rare (< 1%). With the combined MRD information from the first two follow-up time points, it was possible to recognise three different risk groups--55 (43%) were in a low-risk group and had a 3-year relapse rate of only 2% (95% CI 0.05-12%); 19 (15%) were in a high-risk group and had a relapse rate of 75% (55-95%); and 55 (43%) were in an intermediate-risk group and had a 3-year relapse rate of 23% (13-36%). Our collaborative MRD study shows that monitoring patients with childhood ALL at consecutive time points gives clinically relevant insight into the effectiveness of treatment. Combined information on MRD from the first 3 months of treatment distinguishes patients with good prognoses from those with poor prognoses, and this helps in decisions whether and how to modify treatment.

The significance of minimal residual disease in hairy cell leukaemia treated with deoxycoformycin: a long-term follow-up study.

We investigated the clinical significance and long-term follow-up of detecting minimal residual disease (MRD) in hairy cell leukaemia (HCL) in complete remission (CR) after treatment with deoxycoformycin (DCF). MRD was assessed in 23 patients by immunophenotyping peripheral blood and bone marrow frozen sections using a panel of antibodies, CD11c, CD25, CD103 and HC2, which detect hairy cells. 31 cases with active HCL were used as controls. 10/23 patients (43%) had MRD in bone marrow (seven), blood (one) or both sites (two) which was not detected on haematoxylin-eosin bone marrow sections nor by immunohistochemistry on paraffin sections in six cases tested. Sequential studies in four cases did not show changes in the amount of MRD. At a median follow-up of 72 months (range 15-108), 5/23 (22%) patients have relapsed with a median time of 59 months (range 15-105). MRD was detected in three of the five patients who relapsed. In the two patients with negative MRD, one relapsed with an abdominal mass and the other was a late relapse at 84 months. MRD was also documented in 7/18 patients who continued in clinical CR for a median of 80 months (range 63-98). There were no statistical differences in disease-free survival between MRD+ and MRD- patients (P = 0.8). Our findings indicate that relapse after long-term remission achieved with DCF cannot be predicted when MRD is detected by sensitive methods. A search for other parameters such as proliferative rate of the residual cells or chest and abdominal CT scan might identify patients with a higher probability of disease recurrence.