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Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system. Residual leukemic cells after treatment are associated with relapse. Thus, detecting minimal residual disease (MRD) is significant. Major techniques for MRD assessment include multiparameter flow cytometry (MFC), polymerase chain reaction (PCR), and next-generation sequencing (NGS). At a molecular level, AML is the consequence of collaboration of several gene alterations. Some of these gene alterations can also be used as MRD markers to evaluate the level of residual leukemic cells by PCR and NGS. However, when as MRD markers, different gene alterations have different clinical values. This paper aims to summarize the characteristics of various MRD markers, so as to better predict the clinical outcome of AML patients and guide the treatment.

Consolidation chemotherapy in acute myeloid leukemia (AML) aims at eradicating residual leukemic cells and mostly comprises high-dose cytarabine with or without the addition of anthracyclines, including daunorubicin. Immunogenic cell death (ICD) may contribute to the efficacy of anthracyclines in solid cancer, but the impact of ICD in AML is only partly explored. We assessed aspects of ICD, as reflected by calreticulin expression, in primary human AML blasts and observed induction of surface calreticulin upon exposure to daunorubicin but not to cytarabine. We next assessed immune phenotypes in AML patients in complete remission (CR), following consolidation chemotherapy with or without anthracyclines. These patients subsequently received immunotherapy with histamine dihydrochloride (HDC) and IL-2. Patients who had received anthracyclines for consolidation showed enhanced frequencies of CD8

With the awareness of an immunological graft- versus -leukemia (GvL) effect being able to eliminate residual leukemic cells, and, therefore, allowing the use of less intensive conditioning regimens, allogeneic hematopoietic stem cell transplantation (HSCT) is now widely used as a curative therapy

The presence of residual leukemic cells was studied using metaphase-fluorescence in situ hybridization (FISH) in 22 patients with acute myeloid leukemia treated with chemotherapy only or chemotherapy followed by allogeneic bone marrow transplantation. The patients were followed up during their complete remission (CR) for 4 to 108 months (median, 21 months). A total of 88 BM samples was studied. In most of the samples more than 1,000 metaphase cells were analyzed. Residual leukemic cells were detected in 9 of 22 patients (41%). All patients who had an increasing and/or persisting level of abnormal cells in two or more subsequent samples or whose initial samples contained more than 1% of abnormal cells relapsed with one exception, in whom the later subsequent samples showed disappearance of abnormal cells. The time span before the first positive sample seems to be insignificant with regard to the outcome of relapse. Absence or single occurrence of abnormal cells followed by their disappearance was in agreement with CR in all the cases (16 patients). Our results indicate that metaphase-FISH is a reliable tool in the quantitation of residual leukemic cells and provides valuable prognostic information for patients with AML.

Although significant progress has been made in the treatment of the acute leukemias, therapies are generally non-specific and not targeted at the biologic defects underlying these diseases. Consequently, treatment results are suboptimal. The development of leukemic cell phenotype-specific therapies is hampered by our limited knowledge of the biology of acute leukemias. That characterizing the genetic defect may revolutionize treatment approach and disease outcome has recently been proven in acute promyelocytic leukemia. Once identified, genes involved in the pathogenesis of leukemic subtypes not only allow for improved diagnosis and monitoring of minimal residual leukemic cells but may ultimately lead to the development of innovative drug strategies that aim at the inhibition of disease-related genes or their encoded proteins. The focus of this review is to familiarize the practicing physician with some principles of molecular biology and with its current and future goals with respect to leukemia.

The prognostic significance of minimal residual disease (MRD) has been demonstrated for a variety of hematologic malignancies. PCR based assays are among the most important methods for identifying MRD. They are aimed at detecting genetic abnormalities of residual leukemic cells with high specificity and sensitivity and represent an important diagnostic tool to assess the quality of therapeutic response, for clinical risk assessment, and for clinical management. In the present review technical aspects of different MRD detection methods are discussed which depend on the available targets regularly present in the respective leukemia type and subtype. As such fusion transcripts, gene mutations, and clonal rearrangements of antigen-receptor genes may be available for detection. Emphasis is given on discussing benefits and limitations of MRD detection and quantification in CML, AML and ALL.

Autologous (AuSCT) and allogeneic stem cell transplantation (SCT) are well established post-remissional strategies for patients with Acute Myeloid Leukemia (AML). However, there is still a debate ongoing about the relative merit of each of these options in first CR. Minimal residual disease (MRD) may be a useful tool to stratify AML patients into categories of risk which can benefit from differentiated post-remissional approaches. To address this issue, we analysed retrospectively, a series of 123 patients affected with AML in whom flow-cytometry serial determinations of MRD were available, at established time-points (post-induction, post-consolidation, post-stem cell transplantation). All were entered into the EORTC/GIMEMA protocols AML10/AML12 (age 61 yrs), all consisting in intensive induction and consolidation cycles, and, for patients aged the threshold of 3.5x10−4 at post-consolidation check-point is critical to predict disease outcome; MRD− patients have an excellent outcome regardless of the post-consolidation therapy; in the MRD+ group, AuSCT seems not to improve the prognosis whereas the use of SCT is associated with a superior outcome, although a larger number of patients is required to confirm this assumption. Figure Figure

According to the prognostic classifications of karyotypic abnormalities of AML (MRC and SWOG), the intermediate group includes patients either lacking good and poor karyotype or with normal karyotype. Therefore, it represents, by definition, a miscellaneous group for which the evaluation of the better treatment strategy is difficult due to its heterogeneity. Moreover, patients belonging to this intermediate group account for the large majority of AML cases enrolled into clinical trials. The aim of our study was to analyze the factors specifically affecting the outcome of patients bearing intermediate risk karyotypic abnormalities in a group of 94 AML cases entered into the EORTC/GIMEMA protocols AML10/AML12 (age 61 yrs), consisting in intensive induction and consolidation cycles. The clinico-biological variables evaluated in our model included age, FAB, WBC count, MDR1 phenotype, FLT3 mutations and level of post-consolidation bone marrow residual leukemic cells (BMRCL) assessed by multiparametric flow-cytometry (MPFC). In our experience, patients with 2.0x10−4 at the end of consolidation were considered MRDpos and showed a poor prognosis. However, the category of MRDpos patients could be divided into 2 subgroups depending on whether they had a reduction of BMRLC between induction and consolidation, even not reaching the threshold of MRD negativity. In fact, we identified patients whose BMRLC were reduced by at least 1-log between induction and consolidation, referred to as “chemosensitive MRDpos” with an intermediate prognosis, and those showing no reduction of MRD levels between induction and consolidation, referred to as “chemoresistant MRDpos” with the worst outcome. Using the MRC classification, 14/94 patients (15%) had a good-risk cytogenetics, 74/94 (79%) an intermediate-risk and 6/94 (6%) a poor-risk. When we restricted the analysis to cases with intermediate-risk karyotype we found that: 1) in multivariate model, MRD status after consolidation was the only variable significantly affecting relapse free survival (RFS)(P=0.001); 2) patients in the MRDneg, “chemosensitive MRDpos” and “chemoresistant MRDpos” group differed significantly in terms of relapse rate (25% vs. 50% vs. 78%, respectively, P Figure Figure

By multiprametric flow cytometry, we have previously showed that MRD negativity, as defined by a number of BM residual leukemic cells (BMRLC) 61 yrs), all consisting in intensive induction and consolidation cycles, and, for patients aged 2.0×10−4 BMRLC) patients. After induction, 30 (29%) of 105 patients were MRDneg, the remaining 75 (71%) were MRDpos. Fifthy four of 75 (72%) patients in the MRDpos group underwent relapse at a median time of 13 months (range 1–120), while in the MRDneg group 14/30 (47%) had disease relapse at a median time of 21 months (range 4–147) (P=0.014). The 5-year actuarial probability of OS and RFS was 47% and 50%, respectively, for patients in the MRDneg group, compared to 27% and 25%, respectively, for those in the MRDpos group (P=0.031 and 0.008, respectively). Seven patients had early relapse after induction therapy and 98 proceeded to receive consolidation; of these, one died of septic shock and 97 were evaluable for successive MRD assessment. At the post-consolidation time point, 26 (27%) of 97 patiens were MRDneg whereas 71 (73%) were MRDpos. Six of 26 (23%) and 55 of 71 (77%) of patients in MRDneg and in MRDpos group underwent relapse at a median time of 73 months (range 3–147) and 12 months (range 2–120), respectively, (P

In acute myeloid leukemia (AML) patients achieving complete remission, the levels of minimal residual disease (MRD) as determined by flow cytometry have been shown to impact on remission duration and survival. However, some issues such as the most suitable source (BM or PB) or the most appropriate timing (early or delayed evaluation) for MRD determination are still a subject of debate. In our experience, we observed that MRD negativity, as defined by a number of bone marrow residual leukemic cells (BMRLC) 61 yrs), all consisting in intensive induction and consolidation cycles, and allogeneic or autologous stem cell transplantation for patients aged < 61 years. Median age was 53 years (range 17–78), all FAB subtypes were represented with the exception of APL cases which were not included. Eighty-one of 89 patients maintained complete remission after consolidation (8/89 had early relapse after induction) and were suitable for the analysis. After consolidation cycle, 32 of 81 (39%) patients had

I N VITRO c h e m o t h e r a p y in view of e l iminat ing residual leukemic cells f r o m h u m a n autologous graf ts has been invest igated since initial r epo r t of SHARKIS et al. [1] on cyc lophosphamide derivat ives effect on ra t l eukemic cells. Such in v i t ro c h e m o t h e r a p y should be as selective as possible. Unfor tunate ly , due to its cytotoxic mecanism, it is no t so. Therefore , precl inical s tudies had to invest igate the effect of such drugs on l eukemic cells in compar i son to n o r m a l s tem cells in view of es tabl ishing highly efficient t u m o r cell killing while spar ing enough no rm a l s t e m cells to ensure engraftment . Studies rely on two CY derivat ives, 4 Hydroperoxycyclophosphamide and Asta Z 7557, which are; up to now, the only chemother apeu t i c agents used for clinical trials.

We used flow cytometry to quantify minimal residual disease (MRD) in 56 patients with acute myeloid leukemia (AML) expressing a leukemia-associated phenotype. Thirty-four patients aged 18 to 60 years were entered into the AML-10 protocol (induction, consolidation, and autologous stem-cell transplantation [ASCT]), whereas 22 patients older than 60 years received the AML-13 protocol (induction, consolidation, and consolidation II). After induction, the level of MRD that was best associated with treatment outcome was 4.5 × 10−4 residual leukemic cells. However, the outcome in patients with at least 4.5 × 10−4 cells (n = 26) was not significantly different from that in patients with fewer leukemic cells (n = 30); there were 15 (58%) relapses in the first group and 12 (40%) relapses in the second. After consolidation, the most predictive MRD cutoff value was 3.5 × 10−4cells: 22 patients had an MRD level of 3.5 × 10−4 cells or higher and 17 (77%) of these patients had relapse, compared with 5 of 29 patients (17%) with lower MRD levels (P