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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

The tremendous diversity of the human immune repertoire, fundamental for the defense against highly heterogeneous pathogens, is based on the ingenious mechanism of immune gene rearrangements. Rearranged immune genes encoding the immunoglobulins and T-cell receptors and thus determining each lymphocyte’s antigen specificity are very valuable molecular markers for tracing malignant or physiological lymphocytes. One of their most significant applications is tracking residual leukemic cells in patients with lymphoid malignancies. This so called ‘minimal residual disease’ (MRD) has been shown to be the most important prognostic factor across various leukemia subtypes and has therefore been given enormous attention. Despite the current rapid development of the molecular methods, the classical real-time PCR based approach is still being regarded as the standard method for molecular MRD detection due to the cumbersome standardization of the novel approaches currently in progress within the EuroMRD and EuroClonality NGS Consortia. Each of the molecular methods, however, poses certain benefits and it is therefore expectable that none of the methods for MRD detection will clearly prevail over the others in the near future.

Objectives Diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires morphologic expertise; therefore, we evaluated interphase fluorescence in situ hybridization (iFISH) of cerebrospinal fluid (CSF) cytospin preparations as a potential complementary test. Methods Twenty-three CSF cytospin specimens from 13 pediatric patients with ALL were included. iFISH probes detecting BCR-ABL1, ETV6-RUNX1, and KMT2A rearrangement and CDKN2A deletion, which were present at initial diagnosis, were used on follow-up CSF cytospin specimens and were compared with cytology. Results Seventeen (73.9%) follow-up specimens showed concordant results between iFISH and cytology. Two (8.7%) samples with discordant results were positive by iFISH but not by cytology; one (4.3%) was positive only by cytology. In the remaining three (13.0%) specimens, too few cells were available for cytology, whereas iFISH interpretation was possible. Conclusions iFISH of CSF cytospin preparations improves malignant cell detection in pediatric ALL.

Even after microscopic remission after induction therapy, however, in this more than billions of leukemic cells potentially are supposed to escape this microscopic detection. In recent years, technological progress has enabled us to detect residual leukemic cells, which cannot be detected only by means of conventional assessment using microscopic morphology. These residual cells in microscopic remission are “minimal residual diseases (MRD)”. PCR and flow cytometry (FCM) are two major methods to detect MRD, and these methods had several advantages/disadvantages. PCR-MRD can detect as low as 0.001% of residual leukemic cells, while the sensitivity of FCM-MRD is 0.01%. On the other hand, FCM-MRD has advantages in terms of simple preparation methods, short duration to obtain results, cheaper cost, and broad applicability. Irrespective of MRD detection methods, time points to be assessed, and threshold, potent impacts on prognosis have been confirmed by numerous clinical studies. MRD assessment is essential to stratify patients according to relapse risk.

The evaluation of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) has evolved in parallel with the enormous progresses in the therapeutic armamentarium and the application of cutting-edge diagnostic techniques the CLL community witnessed in the past few years. Minimal residual disease is considered an objective measure of disease status defined by the number of residual leukemic cells detected in a sample of peripheral blood and/or bone marrow as proportion of the total white blood cells and defined undetectable if fewer than 1 CLL cell among 10,000 white blood cells (10-4 or 0.01%) is detected. In this review, we aim at shedding light on how to evaluate MRD, what we already know about MRD from the experience with chemoimmunotherapy, and why MRD evaluation remains still relevant in the era of targeted agents.

7513 Background: A goal of AML maintenance therapy is to decrease the risk of relapse by suppressing growth of residual leukemic cells post-induction. CC-486 is an oral hypomethylating agent that allows for extended dosing schedules ( >7 days [d]/28d cycle) to sustain therapeutic activity. In the QUAZAR AML-001 trial (NCT01757535), CC-486 maintenance treatment (Tx) significantly prolonged overall (OS) and relapse-free survival vs. placebo (PBO) in pts with AML in first remission following induction chemotherapy (IC), who were not candidates for hematopoietic stem cell transplant (HSCT). Pts initially received CC-486 or PBO for 14d/cycle, but pts who relapsed with 5–15% blasts could receive escalated 21d/cycle dosing. We review outcomes of pts who received 21d dosing in QUAZAR AML-001. Methods: Pts were aged ≥ 55 years, with intermediate- or poor-risk cytogenetics and ECOG PS ≤ 3, and had achieved first CR/CRi after IC ± consolidation. Within 4 mo of CR/CRi, pts were randomized 1:1 to CC-486 300 mg or PBO QD on d 1–14 of 28d Tx cycles. CR/CRi status was assessed every 3 cycles. Pts relapsing with 5%–15% blasts in blood or bone marrow could receive study drug for 21d/cycle at the investigator’s discretion. Tx could continue until > 15% blasts, unacceptable toxicity, or HSCT. Results: 91 patients (CC-486, 51/238 [21%]; PBO, 40/234 [17%]) were assigned to ≥ 1 21d/cycle dosing schedule. Median time to dose escalation was 9.2 mo (range 1.0-52.7) for CC-486 and 6.0 mo (0.5-19.3) for PBO. Median number of 21d dosing cycles was 2.0 (range 1-45) in the CC-486 arm and 2.0 (1-16) in the PBO arm; 43% and 18% of pts, respectively, received > 3 cycles of 21d dosing. Among 78 evaluable pts, 10/43 (23%) CC-486 pts and 4/35 (11%) PBO pts regained CR/CRi (central review) during dose escalation. Median OS from randomization was 22.8 mo vs. 14.6 mo with CC-486 vs. PBO, respectively (HR 0.66 [95%CI 0.42, 1.0]; P = 0.073), and 1-year survival rates were 80.4% vs. 59.5% (+20.9% [95%CI 2.1%, 39.7%]). The most common AEs with first onset during 21d dosing were febrile neutropenia (CC-486 24%, PBO 3%), thrombocytopenia (22%, 23%), anemia (22%, 20%), and neutropenia (20%, 10%). Conclusions: Escalated 21d CC-486 dosing was well tolerated and resulted in prolongation of OS and restoration of remission in approximately one-fourth of pts. Hematologic AEs first reported during escalated dosing in both Tx arms may be due in part to disease relapse. A 21d dosing schedule should be considered for pts receiving CC-486 who experience relapse with 5–15% blasts. Clinical trial information: NCT01757535 .

Targeting leukemia regenerating cells following chemotherapy can prevent relapse of the disease.

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

To determine the potential of Tc-99m MIBI femoral marrow imaging for detecting minimal residual disease in acute leukemia, MIBI images of 68 patients with acute leukemia and 110 control patients were examined. MIBI accumulation was classified into three patterns: not detectable, mild accumulation, and clearly visualized accumulation. Clearly visualized accumulation was interpreted as abnormal. The mean uptake ratio of the femoral marrow to muscle was calculated. Forty-five patients who were in complete remission (CR) at the time of MIBI imaging had a follow-up study (mean interval, 23 months). Clearly visualized accumulation was demonstrated in 35 patients with acute leukemia: in 7 patients before starting induction chemotherapy, in 12 patients after relapse, and in 16 of the 49 patients in the CR group. Mild accumulation was demonstrated in 14 patients in the CR group and in 13 control group patients. No detectable accumulation was observed in 19 patients in the CR group and in 97 control patients. The marrow and muscle uptake ratio of patients before starting chemotherapy (2.29 +/- 0.26) was greater compared with that in patients after relapse (1.78 +/- 0.44, P0.02) and in patients with abnormal accumulation despite complete remission (1.84 +/- 0.36, P0.01). The uptake ratio in patients with abnormal accumulation despite CR was higher compared with patients with mild accumulation in CR (1.26 +/- 0.13, P0.001) and controls (1.23 +/- 0.10, P0.001) who had mild accumulation. Fifteen patients with abnormal accumulation despite CR had a markedly greater relapse rate (66.7%10.0%, P0.005), a higher mortality rate (46.7%6.7%, P0.01), and shorter remission time (8.7 +/- 10.2 months35.9 +/- 20.1 months, P0.001) compared with 30 patients without abnormal accumulation in CR. MIBI femoral marrow imaging may be a useful and simple method for monitoring levels of residual leukemic cells. Clearly visualized MIBI accumulation may be a marker for relapse.

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.

Procaine and hyperthermia have been shown to possess a relatively selective cytotoxicity to leukemic cells. In this study, the combined effects of procaine and hyperthermia on the growth of hematopoietic progenitors (GM-CFU) and leukemic progenitors (L-CFU) were examined to determine if this combination resulted in a great selective killing of leukemic cells than that achieved by procaine or heat alone. When the cells were treated simutaneously with procaine (2 mM) and hyperthermia (42°C) for one hour, the killing of L-CFU was enhanced considerably whereas GM-CFU were not markedly affected. These data indicate that the combined treatment with procain and hyperthermia might offer an efficient mean to selectively purge residual leukemic cells invitro. Procaine with hyperthermia may have a role in clinical autologous bone marrow transplantation for acute leukemia.

Most of the current strategies in the post-CR treatment of AML favour the use of intensive short-term consolidation followed by early intensification (Heil et al. 1995, Zittoun et al. 1995, Keating et al. 1998, Mitus et al. 1995). Allogeneic bone marrow transplantation (BMT) as early intensification has proved to be superior to autologous BMT or chemotherapy in many experiences, including some controlled trials (Mitus et al. 1995, Zittoun et al. 1997, Zittoun et al. 1995), although autologous BMT seems to offer a limited advantage over chemotherapy in terms of event free survival (EFS) (Zittoun et al. 1995). Furthermore, use of autografting as early intensification makes »ex-vivo« purging advisable in order to minimize the burden of residual leukemic cells in bone marrow harvesting (Gorin 1992), which implies a significant delay in engraftment and an increased risk of transplant-related mortality (Bishop et al. 1997, Damon et al. 1996, Lowenberg 1996). These negative considerations raise the question as to whether early intensification is really the best location for autologous BMT in the treatment of AML.

The maturation and differentiation pathway of normal bone marrow cells can be followed by monoclonal antibodies. Using five-dimensional flow cytometry (forward and side scatter, and three immunofluorescence signals) normal cell maturation is characterized by gradual changes of light-scattering properties and by gradual loss or acquisition of specific cell surface antigens [1–4]. Leukemic blasts can be distinguished from normal bone marrow cells by aberrant expression of surface antigens, e.g., abnormal quantitative expression and coexpression of cell surface antigens not occurring during normal maturation [5–7].

Sixty to seventy percent of adult patients with newly diagnosed AML can achieve a complete remission (CR) through intensive chemotherapy [1–6]. However, the majority of these patients will suffer relapse within 2 years. With the exception of age, secondary leukemias, and a small group of cytogenetically defined AML, no universelly accepted prognostic marker has been identified which would allow early treatment stratification. Thus AML treatment is highly uniform, consisting of intensive induction chemotherapy, followed by consolidating postremission therapy with or without bone marrow transplantation. The “gold standard” for the diagnosis of AML and therapy monitoring is the light microscopic evaluation of cytology and cytochemistry. Its sensitivity for the detection of residual leukemic cells is 5%.

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

We describe a case with acute myelogenous leukemia (AML; M2) who developed prolonged marrow hypoplasia with residual leukemic blasts and recurrent infections after induction chemotherapy. He was treated successfully with a sequential treatment of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and low-dose cytosine arabinoside (LD AraC). To the best of our knowledge this is the first reported case of a successful treatment of a patient with AML, who showed prolonged markedly hypocellular bone marrow with significant residual leukemic cells after induction chemotherapy, with a sequential treatment of GM-CSF and LD AraC.

Bone marrow biopsies obtained from 69 adult patients with acute nonlymphocytic leukemia (ANLL) six to 10 days after initial induction chemotherapy were reviewed blindly to detect the presence of residual leukemia. Discrimination between the presence or absence of leukemic cells was provided by assessment of the numbers, clustering, and nuclear morphology of blasts and promyelocytes. Twenty-six patients had frank leukemia, 25 had no apparent leukemic cells, and 18 had focal residual leukemia. Of 25 patients whose bone marrow contained no detectable residual leukemic cells, 21 gained complete remission without further chemotherapy. These patients had a median duration of remission of 278 days, with five patients still remaining in remission for 578-882 days. Similarly, all of the 18 patients who had focal residual leukemia achieved complete remission without additional chemotherapy; however, all have relapsed with a median duration of remission of 163 days. This study indicates that patients with foci of residual leukemia in their one-week posttreatment bone marrow samples readily achieve remission, but carry a substantial leukemic burden that increases the likelihood of early relapse.

High-dose cytoreductive treatment followed by ABMT represents a new approach to the treatment of acute leukemia as an alternative when leukemic patients do not have HLA-identical donors. ABMT protocol seems to be a valuable treatment for AML if it is immediately employed after the remission obtained to consolidate the remission. For ALL adult patients, and so for poor prognosis ALL in children, intensive therapy with ABMT represents a new approach when conventional therapy has failed or failed to consolidate the remission. The results of ABMT in CML in the literature have been disappointing; the bone marrow could be collected during the course of the first chronic phase after hydroxyurea therapy or other treatment programs. In leukemia the ABMT approach will be more credible if the protocol includes in vitro immunologic or pharmacologic means to eliminate residual leukemic cells.

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