Correlation of minimal residual disease cell frequency with molecular genotype in patients with acute myeloid leukemia.

BACKGROUND: About 70-80 percent of patients with acute myeloid leukemia enter complete remission, but at least half of these patients who achieve remission go on to relapse. Improved treatment is likely to come from increasing the time to relapse, especially for younger patients. With the vastly increasing number of targeted therapies there is a strong need for short-term end-points to efficiently test such therapies for further pursuance. Minimal residual disease assessment may offer such an end-point since it is a strong independent prognostic factor. As proof of principle we examined this concept for FLT3-ITD status at diagnosis. DESIGN AND METHODS: We determined FLT3-ITD status in bone marrow samples from 196 patients with newly diagnosed acute myeloid leukemia. The frequencies of residual leukemic cells of these 196 patients were assessed in 267 follow-up bone marrow samples using immunophenotypic assessment of minimal residual disease. RESULTS: The median frequency of residual leukemic cells after the first cycle of chemotherapy was 8.5-fold higher in patients with FLT3-ITD than in those with wild type FLT3. Such a difference translates into differences in survival, even if other potentially outcome-modulating mutations, such as NPM1, KIT, NRAS, KRAS, FLT3-exon 20 and PTPN11 are included in the analysis. CONCLUSIONS: This study shows that it could be possible to study the efficacy of FLT3 inhibitors using the level of minimal residual disease as a short-term end-point.

High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.

PURPOSE: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34(+)CD38(-) stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse. Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. EXPERIMENTAL DESIGN: First, the leukemogenic potential of unpurified CD34(+)CD38(-) cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34(+)CD38(-) compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. RESULTS: In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34(+)CD38(-) stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34(+) percentage showed no such correlations. CONCLUSIONS: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34(+)CD38(-) population at diagnosis reflects a higher percentage of chemotherapy-resistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.

Immunologic purging of autologous peripheral blood stem cell products based on CD34 and CD133 expression can be effectively and safely applied in half of the acute myeloid leukemia patients.

PURPOSE: Several studies have shown survival benefit by autologous stem cell transplantation in acute myeloid leukemia (AML) after purging of grafts. This has, however, not been confirmed in randomized studies due to high toxicity of purging modalities for normal progenitor/stem cells. In this study, we investigated whether positive selection for CD34+ and/or CD133+ cells, which results in high recovery of normal progenitor/stem cells, is applicable for purging AML grafts. EXPERIMENTAL DESIGN: Positive selections of normal stem cells using CD34 and/or CD133 can be done if one or both markers are absent or have dim expression and remain so during the course of the disease. Marker expressions in newly diagnosed AML were measured with flow cytometry using a cutoff value for positivity of 1%. Stability of marker expression was studied by pairwise comparison of material at diagnosis and relapse. Leukemia associated phenotype expression was used to measure the efficacy of tumor cell reduction. RESULTS: In newly diagnosed AML (n = 165), we found no CD34 and/or CD133 expression in 32% of the cases and dim expression in 20% of the cases. No increase in the percentage of CD34+ cells (n = 44) and CD133+ cells (n = 29) was found in corresponding relapses. Positive selection using grafts contaminated with AML blasts, showing either no or dim expression of CD34 or CD133, resulted in a 3 to 4 log tumor cell reduction (n = 11) with median 50% recovery of normal stem cells. CONCLUSIONS: Purging by positive selection of CD34+ and/or CD133+ cells can safely, effectively, and reproducibly be applied in about 50% of AML cases.

Minimal residual disease in acute myeloid leukemia is predicted by an apoptosis-resistant protein profile at diagnosis.

PURPOSE: Apoptosis is an important mechanism regulating survival of acute myeloid leukemia cells. The apoptosis-related protein profile at diagnosis is important for achieving complete remission thereby affecting survival variables such as disease-free survival (DFS) and overall survival (OS).To investigate the role of the apoptosis protein profile in further response to therapy and outgrowth of disease. EXPERIMENTAL DESIGN: We studied whether Bcl-2, Bcl-xL, Mcl-1, Bax as well as the Bcl-2/Bax ratio and a combination of all (antiapoptosis index, AAI) are related to the frequency of malignant cells surviving the chemotherapy (i.e., minimal residual disease, MRD). MRD cells were identified by leukemia-associated aberrant phenotypes established at diagnosis by flow cytometry. RESULTS: We found that Bcl-2 (R = 0.55, P = 0.002), Bcl-2/Bax (R = 0.42, P = 0.02), and AAI (R = 0.47, P = 0.01) at diagnosis directly correlated with MRD after the first cycle of chemotherapy. In turn, MRD frequency after first cycle correlated with DFS (P = 0.04). Taken together, these results directly explain why Bcl-2/Bax and especially AAI (P = 0.007) at diagnosis correlate with DFS. CONCLUSION: Our results show that apoptosis resistance plays an important role in the first stage of the therapy (i.e., to eliminate the bulk of malignant cells), in terms of achievement of complete remission and frequency of MRD after first cycle of therapy.

C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia.

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34(+)/CD38(-) or CD34(+)/CD33(-) stem cells and present on subsets of CD34(+)/CD38(+) or CD34(+)/CD33(+) progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33(-) AMLs expressed CLL-1. CLL-1 showed variable expression (10-60%) in CD34(+) cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.

Purging of peripheral blood stem cell transplants in AML: a predictive model based on minimal residual disease burden.

OBJECTIVE: Minimal residual disease (MRD) present in peripheral blood stem cell (PBSC) products of AML patients may contribute to relapse. Our goal was to 1) predict leukemia recurrence based on the frequency of MRD present in PBSC products, 2) establish the efficacy of different purging procedures, and 3) integrate this into a model that enables to predict whether or not to purge. METHODS: Minimal residual disease was measured with flow cytometry using leukemia-associated phenotypes as established at diagnosis. Toxicity of purging procedures was established using clonogenic assays. Purging procedures used were cryopreservation, hyperthermia, ether lipid ET-18-OCH3, and combinations. RESULTS: Minimal residual disease in PBSC products correlated significantly with relapse-free survival (n=24, p=0.003). At a cut-off value of 0.05% MRD the relative risk of relapse was 4.6 times lower in the group with less than 0.05% MRD. As measured in 54 PBSC products, the MRD level was less than 0.05% in 17 of 54 cases, between 0.05% and 0.5% in 19 of 54 cases, and higher than 0.5% in 18 of 54 cases. Based on the MRD cut-off of 0.05%, the log tumor reduction needed to achieve this threshold is zero for the 17 of 54 cases in which MRD was below 0.05%, less than or equal to 1 log in 19 of 54 cases, and greater than 1-2 log in 18 of 54 cases. When applying purging with 25 mug/mL ET-18-OCH3 combined with cryopreservation at 10% DMSO and hyperthermia at 42 degrees C combined with cryopreservation at 10% or 4% DMSO, there was greater than or equal to 1 log depletion of AML blasts. CONCLUSION: This study establishes (1) a threshold level for MRD above which prognosis is worse, (2) that stem cell products from 69% of patients have higher than this "safe" MRD level, and (3) that ET-18-OCH3 and hyperthermia may be used to purge products in part of these patients.

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

BACKGROUND AND OBJECTIVES: The percentages of CD34+ cells in the bone marrow of patients with acute myeloid leukemia (AML) vary widely. Especially in the low range (<5% CD34+ cells), the nature (normal or malignant) of the CD34+ cells is uncertain. Since only in a minority of cases are molecular techniques applicable, in this study we explored a multiparameter approach using phenotypic and functional characteristics to discriminate normal CD34+ cells from malignant ones. DESIGN AND METHODS: CD34+ cells from 24 AML patients with <5% CD34+ cells and from 3 patients with >50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants.

High class II-associated invariant chain peptide expression on residual leukemic cells is associated with increased relapse risk in acute myeloid leukemia.

The presence of class II-associated invariant chain (CLIP) on leukemic cells is negatively associated with clinical outcome in untreated acute myeloid leukemia (AML). CLIP plays a role in the immune escape of leukemic cells, suggesting that it impairs the immunogenicity of minimal residual disease (MRD) cells causing a relapse. Here, we demonstrate that CLIP expression on leukemia-associated phenotype (LAP)-positive cells during follow-up is significantly correlated with a shortened relapse-free survival, even in those patients who are generally considered as MRD(low) (0.01-0.1% LAP(+) cells). Consequently, CLIP evaluation could be of additional value in the evaluation of MRD to predict a relapse of AML.

Interleukin-2 receptor alpha-chain (CD25) expression on leukaemic blasts is predictive for outcome and level of residual disease in AML.

We investigated the role of CD25 as a prognostic marker in acute myeloid leukaemia (AML). Seventy-two newly diagnosed patients < or =60 years were retrospectively analysed by flow cytometry for CD25 positivity of AML blasts. Patients with CD25 expression of >10%, when compared to < or =10%, had a significantly shorter overall survival (OS, p=0.0005) and relapse-free survival (RFS, p=0.005). In multivariate analysis CD25 expression is an independent adverse factor for OS and RFS. High CD25 combined with FLT3-ITD positivity resulted in the poorest OS and RFS (p=0.001 and p=0.003, respectively). CD25 expression remained prognostic within the intermediate cytogenetic risk group. In addition, after the first cycle of chemotherapy, a significantly higher MRD frequency was found in patients expressing CD25 above cut-off (p=0.003). Our results show that CD25 expression is an independent adverse prognostic marker in AML patients < or =60 and correlates with MRD.

[Guideline on urothelial carcinoma of the bladder]. / Richtlijn urotheelcarcinoom van de blaas.

Urothelial carcinoma of the bladder is diagnosed predominantly in people over 60 years of age. The most common symptom is haematuria. Smoking is an important risk factor (relative risk 2.5 to 3). Cystoscopy is performed whenever bladder carcinoma is suspected. The recurrence rate of a non-muscle invasive urothelial carcinoma is high (31-78% within 5 years). A single intravesical instillation with a chemotherapeutic agent within 24 hours of transurethral resection (TUR) reduces the risk of recurrence. Carcinoma in situ (CIS) should be treated as high-grade urothelial carcinoma. Standard treatment for patients with non-metastasized muscle-invasive urothelial carcinoma is cystectomy in combination with extensive lymph node dissection. There are several possibilities for urinary diversion following cystectomy, none of which are any better than the others. Bladder-sparing brachytherapy may be used in patients with solitary T1 - T2 urothelial carcinoma < 5 cm. Neoadjuvant cisplatin-containing chemotherapy prior to cystectomy in muscle-invasive carcinoma only slightly improves survival. Cisplatin-containing combination chemotherapy is the standard treatment for metastasized urothelial carcinoma.

Positive selection for CD90 as a purging option in acute myeloid leukemia stem cell transplants.

BACKGROUND: Several studies showed the benefit of purging of acute myeloid leukemia (AML) stem cell transplants. We reported previously that purging by positive selection of CD34+ and CD133+ cells resulted in a 3-4 log tumor cell reduction (TCR) in CD34- and/or CD133- AML, but has been shown to be potentially applicable in only about 50% of cases. Similar to CD34 and CD133, CD90 marks the hematopoietic CD34 positive stem cells capable of full hematopoietic recovery after myeloablative chemotherapy, and therefore, in the present study, we explored whether a similar purging approach is possible using CD90. METHODS: CD90 expression was established by flowcytometry in diagnosis AML on the clonogenic AML CD34+ blast population by flow cytometry. Positivity was defined as >3% CD90 (CD34+) expression on blasts. For the calculation of the efficacy of TCR by positive selection, AML blasts were recognized by either prelabeling diagnosis blasts with CD45-FITC in spiking model experiments or using expression of leukemia associated marker combinations both in spiking experiments and in real transplants. RESULTS: In 119 patients with AML and myelodysplastic syndrome, we found coexpression of CD34 and CD90 (>3%) in 42 cases (35%). In AML patients 60 years or younger, representing the patients who are eligible for transplantation, only 23% (16/69) of the patients showed CD90 expression. Positive selection for CD90 in transplants containing CD90 negative AML resulted in a 2.8-4 log TCR in the models used. CONCLUSIONS: Purging by positive selection using CD90 can potentially be applied effectively in the majority of AML patients 60 years or younger.

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

Function of the ABC transporters, P-glycoprotein, multidrug resistance protein and breast cancer resistance protein, in minimal residual disease in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Relapse is common in acute myeloid leukemia (AML) because of persistence of minimal residual disease (MRD). ABC-transporters P-glycoprotein (Pgp) and multidrug resistance protein (MRP), are thought to contribute to treatment failure, while it is unknown whether breast cancer resistance protein (BCRP) does so. However, whether up-regulation of pump activity or selection of subpopulations with higher pump activity occurs during chemotherapy is unclear. The aim of this study was to elucidate whether ABC-transporter function changes during the course of disease. DESIGN AND METHODS: MRD cells were identified using leukemia-associated phenotypes combined with a fluorescent probe assay with substrate/modulator: Syto16/ PSC833 (Pgp), calcein-AM/probenecid (MRP) and BODIPY-prazosin/Ko143 (BCRP); efflux profiles were directly compared with blasts at diagnosis and relapse from the same patient. RESULTS: At diagnosis BCRP activity was undetectable in AML blasts from 23/26 cases, while Pgp activity was present in 36/45 and MRP activity in 26/44 of the cases. Furthermore, no subpopulations of blasts with considerably higher drug efflux capacities were found. Overall, no consistent changes were observed at follow-up [during chemotherapy (n=20), MRD (n=37), relapse (n=26))] in forty-five patients, the mean activities (as percentages of values at diagnosis) were 97% (Pgp), 103% (MRP) and 102% (BCRP). INTERPRETATION AND CONCLUSIONS: Emergence of MRD is thus not accompanied by either upregulation of ABC-transporter function during or after chemotherapy or by selection of pre-existing highly resistant subpopulations. The prognostic value of Pgp and MRP is, therefore, likely related to drug efflux capacity homogeneously distributed in the whole blast population, while BCRP probably has a limited function in drug efflux-related resistance in AML.