Next-generation sequencing for measurable residual disease detection in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a blood cancer characterized by acquired genetic mutations. There is great interest in accurately establishing measurable residual disease (MRD) burden in AML patients in remission after treatment but at risk of relapse. However, inter- and intrapatient genetic diversity means that, unlike in the chronic myeloid and acute promyelocytic leukaemias, no single genetic abnormality is pathognomonic for all cases of AML MRD. Next-generation sequencing offers the opportunity to test broadly and deeply for potential genetic evidence of residual AML, and while not currently accepted for such use clinically, is likely to be increasingly used for AML MRD testing in the future.     

Minimal residual disease directed therapy for childhood acute myeloid leukaemia: the time is now

The continued improvement in the prognosis of childhood acute myeloid leukaemia (AML) has been paralleled by the use of increasingly intensive therapy. This has led to attempts to develop risk-directed strategies in which the most intensive treatment is reserved for those at highest risk of relapse. Unfortunately, current approaches, which rely on cytogenetic sub-grouping and morphological assessment of response to therapy, are inaccurate. New prognostic factors are needed. This annotation proposes that the introduction of protocols based on the measurement of minimal residual disease (MRD) holds the key to progression from an era of 'cure at all costs' to a more individualised approach. However, the full potential of MRD technologies will only be realised through properly designed studies with scrupulous attention to logistics and quality assurance. The article illustrates which children may benefit most from MRD analysis in AML and explores practical issues that should be addressed in the design of clinical trials.     

Clinical significance of minimal residual disease in patients with acute leukaemia undergoing haematopoietic stem cell transplantation

In patients with acute leukaemia, the relative risk of relapse influences the choice between chemotherapy and haematopoietic stem cell transplantation (HSCT). The demonstration that minimal residual disease (MRD) is the strongest overall prognostic indicator and can identify patients who are unlikely to be cured by standard chemotherapy has added a powerful new factor to consider when making this decision. There is substantial data indicating that the likelihood of relapse after transplant is directly correlated with levels of MRD before transplant. This knowledge can be used to adjust the timing of HSCT, and guide the selection of donor, conditioning regimen, and post‐HSCT strategies to maximize the graft‐versus‐leukaemia effect. Because MRD emerging post‐transplant carries a dire prognosis, its detection can trigger withdrawal of immunosuppression, additional cellular and molecular therapies, or preparations for a second HSCT. Although it is not yet clear whether any of these actions will significantly improve outcome, it is likely that they will be most effective for patients with a relatively low tumour burden, who can be identified only through MRD testing. In this article, we review the clinical significance of MRD in the context of autologous and allogeneic HSCT.     

Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia

In children with acute myeloid leukaemia (AML), morphological and karyotypic studies cannot precisely assess response to treatment, and less than one-third of patients have genetic markers for molecular studies of residual disease. We determined the usefulness of a four-colour flow cytometric strategy developed in our laboratory to study residual disease. We first compared the immunophenotypes of AML cells obtained from 54 children at diagnosis with those of cells from 59 normal or regenerating bone marrow samples. Forty-six of the 54 AML cases (85.2%) had immunophenotypes that allowed detection of 0.1-0.01% residual leukaemic cells. Of 230 bone marrow samples obtained from those 46 patients during and off treatment, 61 (26.5%) had >/= 0.1% AML cells by flow cytometry. We found that core binding factor-associated AML had a significantly better early treatment response. Mean (+/- standard error) 2-year survival estimate was 33.1 +/- 19.1% for patients with >/= 0.1% AML cells by flow cytometry after induction therapy, but 72.1 +/- 11.5% for those with < 0.1% AML cells (P = 0.022); overt recurrence of AML within the subsequent 6 months was significantly more likely in the former group. The assay described here holds promise for guiding the choice of post-remission treatment options in children with AML.     

Minimal residual disease detection after allogeneic stem cell transplantation is correlated to relapse in patients with acute lymphoblastic leukaemia

Minimal residual disease (MRD) assessments were performed retrospectively after allogeneic stem cell transplantation (SCT) in 32 patients (23 children and nine adults) with acute lymphoblastic leukaemia (ALL). Using immunoglobulin and T-cell receptor rearrangements as clonal markers, MRD was detected after SCT in nine patients, eight of whom have relapsed. The median time between first MRD detection and relapse was 5.5 (range 0.5-30) months. In 23 patients without MRD, six have relapsed to date: lower sensitivity, central nervous system relapse and clonal exchange of the leukaemic clone were factors that may explain the failure to detect MRD before relapse in these patients. In univariate analysis, factors associated with decreased risk of relapse were transplantation in first remission (P=0.02), the combination of acute and chronic graft-versus-host disease (P=0.03) and absence of MRD after SCT (P=0.005). In multivariate analysis, only MRD detection after SCT was significantly associated with increased risk of relapse (P=0.05). In conclusion, MRD detection after SCT is correlated with relapse and provides the opportunity for initiating immunotherapeutic intervention at an early stage when the tumour cell burden is still low.     

Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia

There is little information available regarding immunophenotypic monitoring of minimal residual disease (MRD) in acute myeloid leukaemia (AML). We investigated leukaemic cells co‐expressing CD15 and CD117 (CD15+CD117+) in 72 adult AML cases at diagnosis. In 22 cases (31%) with various AML subtypes, more than 5% of leukaemic cells showed the CD15+CD117+ phenotype (range 5.22–55.48%). These 22 cases were younger and had a higher complete remission (CR) rate than the other AML cases, but the CD15+CD117+ cell percentage at diagnosis showed no correlation with the CR duration among the 72 cases. The CD15+CD117+ cell percentage showed a range of 0.00–0.08% in bone marrow cells from 10 haematologically normal subjects. We also investigated CD15+CD117+ cells in sequential bone marrow samples from 17 AML patients who achieved CR and who had had more than 5% CD15+CD117+ leukaemic cells at diagnosis. Because the CD15+CD117+ cell percentage varied among these AML cases, we calculated the percentage of MRD {MRD% = [CD15+CD117+ cells (%) in each sequential marrow sample] ÷ [CD15+CD117+ cells (%) at diagnosis of the corresponding case] × 100}. A high MRD% after 10 months of CR was significantly associated with a short CR duration (P = 0.0004), whereas continuation of a well‐reduced MRD% was associated with a long CR duration. The leukaemic cells conserved the CD15+CD117+ phenotype in all of the eight cases who relapsed. Flow cytometric monitoring of CD15+CD117+ cells is simple and can be applied to a substantial fraction of AML cases. This monitoring may be useful for predicting relapse of adult AML.     

Abemaciclib drives the therapeutic differentiation of acute myeloid leukaemia stem cells

Self-renewal and differentiation arrest are two features of leukaemia stem cells (LSCs) responsible for the high relapse rate of acute myeloid leukaemia (AML). To screen drugs to overcome differentiation blockade for AML, we conducted screening of 2040 small molecules from a library of United States Food and Drug Administration-approved drugs and found that the cyclin-dependent kinase (CDK)4/6 inhibitor, abemaciclib, exerts high anti-leukaemic activity. Abemaciclib significantly suppressed proliferation and promoted the differentiation of LSCs in vitro. Abemaciclib also efficiently induced differentiation and impaired self-renewal of LSCs, thus reducing the leukaemic cell burden and improving survival in various preclinical animal models, including patient-derived xenografts. Importantly, abemaciclib strongly enhanced anti-tumour effects in combination with venetoclax, a B-cell lymphoma 2 (Bcl-2) inhibitor. This treatment combination led to a marked decrease in LSC-enriched populations and resulted in a synergistic anti-leukaemic effect. Target screening revealed that in addition to CDK4/6, abemaciclib bound to and inhibited CDK9, consequently attenuating the protein levels of global p-Ser2 RNA Polymerase II (Pol II) carboxy terminal domain (CTD), Myc, Bcl-2, and myeloid cell leukaemia-1 (Mcl-1), which was important for the anti-AML effect of abemaciclib. Collectively, these data provide a strong rationale for the clinical evaluation of abemaciclib to induce LSC differentiation and treat highly aggressive AML as well as other advanced haematological malignancies.     

Delayed Haematological recovery after autologous stem cell transplantation is associated with favourable outcome in acute myeloid leukaemia

Autologous stem cell transplantation (ASCT) is applied to consolidate first remission in patients with acute myeloid leukaemia (AML). However, outcome after ASCT widely varies among AML patients. We analyzed the prognostic significance of haematological recovery for neutrophils [absolute neutrophil count (ANC) >1·0 × 10⁹/l] and platelets (platelet count >20·0 × 10⁹/l), stratifying at day 20 after ASCT in 88 consecutive and homogeneously treated AML patients in first remission. We observed that patients with delayed recovery had better overall survival (OS; ANC: P < 0·0001 and platelets: P = 0·0062) and time to progression (TTP; ANC: P = 0·0003 and platelets: P = 0·0125). Delayed recovery was an independent marker for better OS and TTP in a multivariate analysis including age, gender, number of transfused CD34+ cells, cytogenetics, FLT3‐internal tandem duplication and NPM1 mutation. Our results suggest that delayed neutrophil and platelet recovery is associated with longer OS and TTP in AML patients consolidated with ASCT in first remission.     

Autologous stem cell transplantation in the treatment of adults with acute myeloid leukaemia

Most adult patients under 60 years with acute myeloid leukaemia (AML) who achieve a complete remission after induction chemotherapy will relapse if they do not receive further therapy. Consolidation treatment with autologous stem cell transplantation (SCT) is one option that has been studied extensively. High-dose cytotoxic therapy followed by autologous SCT or intensive cycles of chemotherapy furnish overall approximately similar probabilities of survival when applied in first remission. Here, we present a concise update regarding the place of autologous SCT in the treatment of AML. Particular issues discussed are the value of autologous SCT in different prognostic subsets of AML and the value of autologous mobilised peripheral blood stem cell transplants, which offer a much faster haematopoietic recovery.     

Phase I/II study of vaccination with dendritic-like leukaemia cells for the immunotherapy of acute myeloid leukaemia

Twenty-two patients with acute myeloid leukaemia were recruited into a phase I/II clinical trial investigating the vaccination of patients in complete remission (CR) with autologous dendritic-like leukaemia cells (DLLC). At trial entry, leukaemia cells were harvested and tested for their ability to undergo cytokine-induced dendritic cell differentiation. Patients were then treated with intensive chemotherapy. Five patients achieved both CR and had leukaemia cells that successfully underwent differentiation and therefore proceeded to vaccination. Four escalating doses of DLLC were administered weekly by subcutaneous injection. Vaccination was generally well tolerated although one patient developed extensive eczema and an increased antinuclear factor titre possibly indicating induction of autoimmunity. Development of anti-leukaemic T-cell responses was assessed by enzyme-linked immunospot analysis of gamma-interferon secreting T lymphocytes and by human leucocyte antigen tetramer analysis for WT1-specific T cells. Increases in anti-leukaemic T-cell responses were demonstrated in four patients, but only two of the five remained in remission more than 12 months postvaccination. The study has demonstrated that generation of DLLC is feasible in only a subgroup of patients and is currently neither broadly applicable or clinically effective.     

Minimal residual disease assessed by multi‐parameter flow cytometry is highly prognostic in adult patients with acute lymphoblastic leukaemia

The prognostic value of minimal residual disease (MRD) assessed by multi‐parameter flow cytometry (MFC) was investigated among 340 adult patients with B‐cell acute lymphoblastic leukaemia (B‐ALL) treated between 2004 and 2014 using regimens including the hyperCVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine) backbone. Among them, 323 (95%) achieved complete remission (CR) and were included in this study. Median age was 52 years (range, 15–84). Median white blood cell count (WBC) was 9·35 × 10⁹/l (range, 0·4–658·1 ×1 0⁹/l). MRD by MFC was initially assessed with a sensitivity of 0·01%, using a 15‐marker, 4‐colour panel and subsequently a 6‐colour panel on bone marrow specimens obtained at CR achievement and at approximately 3 month intervals thereafter. MRD negative status at CR was associated with improved disease‐free survival (DFS) and overall survival (OS) (P = 0·004 and P = 0·03, respectively). Similarly, achieving MRD negative status at approximately 3 and 6 months was associated with improved DFS (P = 0·004 and P < 0·0001, respectively) and OS (P = 0·004 and P < 0·0001, respectively). Multivariate analysis including age, WBC at presentation, cytogenetics (standard versus high risk) and MRD status at CR, 3 and 6 months, indicated that MRD negative status at CR was an independent predictor of DFS (P < 0·05). Achievement of an MRD negative state assessed by MFC is an important predictor of DFS and OS in adult patients with ALL.     

Absence of leukaemic CD34+ cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered

Primary resistance and relapses after initial successful treatment are common in acute myeloid leukaemia and therefore outcome remains poor. More accurate risk group stratification and effective personalized risk adapted treatment are necessary to improve outcome. In the last two decades, controversial results have been published concerning the prognostic relevance of CD34 expression. In this study of 706 acute myeloid leukaemia patients, we established a new flow cytometric‐based CD34‐definition, without use of cut‐off values. We discriminated CD34‐positive (n = 548) and CD34‐negative (n = 158) patients by the presence or absence of neoplastic CD34+ cells, respectively. CD34‐status was defined using aberrant immunophenotypes and validated using molecular phenotypes. This new definition of CD34 enables strong prediction of treatment outcome in the entire patient group and in several risk subgroups. Previously observed discrepancies in prognostic impact of CD34 protein expression using cut‐offs (5–20%) can now entirely be explained by considering the number of CD34‐negative cases. In the total patient group, the absence of neoplastic CD34‐positive cells is paralleled by low levels of minimal residual disease, suggesting relative therapy sensitivity and explaining longer survival. Overall, we present CD34 surface expression as a relatively simple, powerful and independent predictor of clinical outcome, now warranting incorporation in acute myeloid leukaemia risk stratification.     

Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia: a metaphase-FISH study

Metaphase-FISH was adopted for the detection of proliferating Philadelphia-positive (Ph⁺) residual leukaemic cells in 25 patients with chronic myeloid leukaemia treated with allogeneic bone marrow transplantation (BMT). Patients were followed up during their clinical remission for 4–50 months (median 17 months) after BMT. 80 bone marrow samples were studied. For most of the cases no fewer than 1000 metaphases were analysed. Six patients (24%) showed residual Ph⁺ cells during the first 6 months and two others by the end of the first year after BMT. Three patients relapsed during the study and in two of them residual Ph⁺ cells were detected during the first 6 months after BMT. In 17 patients no Ph⁺ cells were detected at any stage of follow-up and 16 (94.1%) of them continue in complete clinical and haematological remission. Our results indicate that metaphase-FISH is a reliable tool in the quantitation of proliferating residual leukaemic cells. We suggest that consecutive findings of equal amounts of residual leukaemic cells do not necessarily predict a relapse. However, their presence calls for follow-up at shorter intervals where an increasing number of these cells predicts an ensuing relapse.     

High-dose idarubicin and busulphan as conditioning to autologous stem cell transplantation in adult patients with acute myeloid leukaemia

Between 30 and 50% of patients with acute myeloid leukaemia (AML) relapse after autologous stem cell transplantation (ASCT). One possibility of reducing the relapse rate could be the adoption of conditioning regimens specifically designed for AML. We report treatment results achieved with a new conditioning for ASCT, based on high-dose idarubicin (IDA) plus oral busulphan. Patients (n = 40) were conditioned with a regimen consisting of 3 d continuous intravenous infusion IDA at 20 mg/m2, followed by 4 d conventional dose oral busulphan. Unpurged peripheral blood stem cells were used in all cases. All patients had non-M3-AML and were in first complete remission (CR). The median number of CD34+ cells infused was 6.9 x 10(6)/l (2.6-24). No case of transplant-related mortality occurred. In all cases, left ventricular ejection fraction remained unmodified after ASCT. Thirty-three of 40 patients (82%) had grade 3-4 mucositis requiring total parenteral nutrition in all cases. After a median follow up for surviving patients of 32 months from ASCT, 30 patients (75%) are alive and 26 (65%) are in continuous CR. Our data show that a conditioning regimen based on high-dose IDA plus busulphan results in an encouraging reduction of the relapse rate after ASCT in AML.     

The role of allogeneic stem cell transplantation in the management of acute myeloid leukaemia: a triumph of hope and experience

Acute myeloid leukaemia (AML) is the commonest indication for allogeneic stem cell transplantation (allo-SCT) worldwide. The accumulated experience of allografting in AML over the last four decades has provided critical insights into both the contribution of the conditioning regimen and the graft-versus-leukaemia effect to the curative potential of the most common form of immunotherapy utilised in standard clinical practice. Coupled with advances in donor availability and transplant technologies, this has resulted in allo-SCT becoming an important treatment modality for the majority of adults with high-risk AML. At the same time, advances in genomic classification, coupled with progress in the accurate quantification of measurable residual disease, have increased the precision with which allo-mandatory patients can be identified, whilst simultaneously permitting accurate identification of those patients who can be spared the toxicity of an allograft. Despite this progress, disease recurrence still remains a major cause of transplant failure and AML has served as a paradigm for the development of strategies to reduce the risk of relapse ‒ notably the novel concept of post-transplant maintenance, utilising pharmacological or cellular therapies.     

Feasibility of BAALC gene expression for detection of minimal residual disease and risk stratification in normal karyotype acute myeloid leukaemia

High BAALC gene expression has been associated with poor prognosis in cytogenetically normal acute myeloid leukaemia (CN‐AML) and has been suggested as a suitable marker for assessing minimal residual disease (MRD). The purpose of this study was to substantiate these findings by the analysis of a large data set of 632 diagnostic and follow‐up samples in 142 intensively treated CN‐AML patients. Paired diagnostic/relapse samples of 35 patients revealed stable high BAALC expression in 89%, irrespective of a high proportion of clonal evolution found in 49% of these cases. High BAALC expression, both directly after induction chemotherapy and within 3–6 months after induction chemotherapy, correlated significantly with shorter event‐free survival and overall survival. Moreover, 8 of 10 patients displaying high BAALC expression levels after completion of induction therapy as well as 5 of 5 patients exhibiting high BAALC expression levels within 3–6 months after induction chemotherapy experienced relapse with a median of 197 and 101 days, respectively, from sampling to relapse. Thus, BAALC expression‐based MRD detection during therapy may be considered a strategy to identify patients at high risk of relapse.