Sylvie Freeman

Addition of Gemtuzumab Ozogamicin to Induction Chemotherapy Improves Survival in Older Patients With Acute Myeloid Leukemia

PURPOSE There has been little survival improvement in older patients with acute myeloid leukemia (AML) in the last two decades. Improving induction treatment may improve the rate and quality of remission and consequently survival. In our previous trial, in younger patients, we showed improved survival for the majority of patients when adding gemtuzumab ozogamicin (GO) to induction chemotherapy. PATIENTS AND METHODS Untreated patients with AML or high-risk myelodysplastic syndrome (median age, 67 years; range, 51 to 84 years) were randomly assigned to receive induction chemotherapy with either daunorubicin/ara-C or daunorubicin/clofarabine, with (n = 559) or without (n = 556) GO 3 mg/m(2) on day 1 of course one of therapy. The primary end point was overall survival (OS). RESULTS The overall response rate was 69% (complete remission [CR], 60%; CR with incomplete recovery [CRi], 9%), with no difference between GO (70%) and no GO (68%) arms. There was no difference in 30- or 60-day mortality and no major increase in toxicity with GO. With median follow-up of 30 months (range, 5.5 to 54.6 months), 3-year cumulative incidence of relapse was significantly lower with GO (68% v 76%; hazard ratio [HR], 0.78; 95% CI, 0.66 to 0.93; P = .007), and 3-year survival was significantly better (25% v 20%; HR, 0.87; 95% CI, 0.76 to 1.00; P = .05). The benefit was apparent across subgroups. There was no interaction with other treatment interventions. A meta-analysis of 2,228 patients in two United Kingdom National Cancer Research Institute trials showed significant improvements in relapse (HR, 0.82; 95% CI, 0.72 to 0.93; P = .002) and OS (HR, 0.88; 95% CI, 0.79 to 0.98; P = .02). CONCLUSION Adding GO (3 mg/m(2)) to induction chemotherapy reduces relapse risk and improves survival with little increase in toxicity.

Assessment of Minimal Residual Disease in Standard-Risk AML.

BACKGROUND Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODS We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTS Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONS The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

The Myeloid-specific Sialic Acid-binding Receptor, CD33, Associates with the Protein-tyrosine Phosphatases, SHP-1 and SHP-2

The myeloid restricted membrane glycoprotein, CD33, is a member of the recently characterized “sialic acid-binding immunoglobulin-related lectin” family. Although CD33 can mediate sialic acid-dependent cell interactions as a recombinant protein, its function in myeloid cells has yet to be determined. Since CD33 contains two potential immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail, we investigated whether it might act as a signaling receptor in myeloid cells. Tyrosine phosphorylation of CD33 in myeloid cell lines was stimulated by cell surface cross-linking or by pervanadate, and inhibited by PP2, a specific inhibitor of Src family tyrosine kinases. Phosphorylated CD33 recruited both the protein-tyrosine phosphatases, SHP-1 and SHP-2. CD33 was dephosphorylated in vitro by the co-immunoprecipitated tyrosine phosphatases, suggesting that it might also be an in vivo substrate. The first CD33 phosphotyrosine motif is dominant in CD33-SHP-1/SHP-2 interactions, since mutating tyrosine 340 in a CD33-cytoplasmic tail fusion protein significantly reduced binding to SHP-1 and SHP-2 in THP-1 lysates, while mutation of tyrosine 358 had no effect. Furthermore, the NH2-terminal Src homology 2 domain of SHP-1 and SHP-2, believed to be essential for phosphatase activation, selectively bound a CD33 phosphopeptide containing tyrosine 340 but not one containing tyrosine 358. Finally, mutation of tyrosine 340 increased red blood cell binding by CD33 expressed in COS cells. Hence, CD33 signaling through selective recruitment of SHP-1/SHP-2 may modulate its ligand(s) binding activity.

Prognostic Relevance of Treatment Response Measured by Flow Cytometric Residual Disease Detection in Older Patients With Acute Myeloid Leukemia

PURPOSE Older patients with acute myeloid leukemia (AML) have a high relapse rate after standard chemotherapy. We investigated whether measuring chemotherapy sensitivity by multiparameter flow cytometric minimal residual disease (MFC-MRD) detection has prognostic value in patients older than age 60 years or is simply a surrogate for known age-related risk factors. PATIENT AND METHODS Eight hundred ninety-two unselected patients treated intensively in the United Kingdom National Cancer Research Institute AML16 Trial were assessed prospectively for MFC-MRD during treatment. Eight hundred thirty-three patients had leukemia-associated immunophenotypes (LAIPs) identified by pretreatment screening. Four hundred twenty-seven patients entered complete remission (CR) after one or two courses (designated C1 and C2, respectively) and were MFC-MRD assessable by LAIP detection in CR bone marrow for at least one of these time points. MRD positivity was defined as residual disease detectable by LAIP. RESULTS MFC-MRD negativity, which was achieved in 51% of patients after C1 (n = 286) and 64% of patients after C2 (n = 279), conferred significantly better 3-year survival from CR (C1: 42% v 26% in MRD-positive patients, P < .001; C2: 38% v 18%, respectively; P < .001) and reduced relapse (C1: 71% v 83% in MRD-positive patients, P < .001; C2: 79% v 91%, respectively; P < .001), with higher risk of early relapse in MRD-positive patients (median time to relapse, 8.5 v 17.1 months, respectively). In multivariable analysis, MRD status at the post-C1 time point independently predicted survival, identifying a subgroup of intermediate-risk patients with particularly poor outcome. However, survival benefit from gemtuzumab ozogamicin was not associated with MFC-MRD chemotherapy sensitivity. CONCLUSION Early assessment of treatment response using flow cytometry provides powerful independent prognostic information in older adults with AML, lending support to the incorporation of MRD detection to refine risk stratification and inform clinical trial design in this challenging group of patients.

Sialoadhesin binds preferentially to cells of the granulocytic lineage.

Sialoadhesin is a macrophage-restricted, sialic acid-dependent receptor of 185 kD that binds to the oligosaccharide sequence NeuAc alpha 2,3Gal on cell surface glycoconjugates. Recent cDNA cloning has shown that sialoadhesin is a new member of the immunoglobulin superfamily with sequence similarity to CD22, a sialic acid-dependent receptor of B lymphocytes. Sialoadhesin has been implicated in cellular interactions of stromal macrophages with developing myeloid cells. In this study, direct evidence for this interaction was obtained in cell-cell binding assays using both native and recombinant forms of the protein. In all assays, sialoadhesin exhibited specific, differential binding to various murine cell populations of hemopoietic origin. In rank order, sialoadhesin bound neutrophils > bone marrow cells = blood leukocytes > lymphocytes > thymocytes. Single-cell analyses confirmed that sialoadhesin selectively bound myeloid cells in complex cell mixtures obtained from the bone marrow and blood. In comparison, a recombinant Fc-chimeric form of murine CD22 showed high binding to B and T lymphocytes, but very low binding to immature and mature myeloid cells. These results are consistent with the notion that sialoadhesin in involved in interactions with granulocytes at different stages of their life histories.

Multicenter validation of a reproducible flow cytometric score for the diagnosis of low-grade myelodysplastic syndromes: results of a European LeukemiaNET study

Background The current World Health Organization classification of myelodysplastic syndromes is based morphological evaluation of bone marrow dysplasia. In clinical practice, the reproducibility of the recognition of dysplasia is usually poor especially in cases that lack specific markers such as ring sideroblasts and clonal cytogenetic abnormalities. Design and Methods We aimed to develop and validate a flow cytometric score for the diagnosis of myelodysplastic syndrome. Four reproducible parameters were analyzed: CD34+ myeloblast-related and B-progenitor-related cluster size (defined by CD45 expression and side scatter characteristics CD34+ marrow cells), myeloblast CD45 expression and granulocyte side scatter value. The study comprised a “learning cohort” (n=538) to define the score and a “validation cohort” (n=259) to confirm its diagnostic value. Results With respect to non-clonal cytopenias, patients with myelodysplastic syndrome had increased myeloblast-related cluster size, decreased B-progenitor-related cluster size, aberrant CD45 expression and reduced granulocyte side scatter (P<0.001). To define the flow cytometric score, these four parameters were combined in a regression model and the weight for each variable was estimated based on coefficients from that model. In the learning cohort a correct diagnosis of myelodysplastic syndrome was formulated in 198/281 cases (sensitivity 70%), while 18 false-positive results were noted among 257 controls (specificity 93%). Sixty-five percent of patients without specific markers of dysplasia (ring sideroblasts and clonal cytogenetic abnormalities) were correctly classified. A high value of the flow cytometric score was associated with multilineage dysplasia (P=0.001), transfusion dependency (P=0.02), and poor-risk cytogenetics (P=0.04). The sensitivity and specificity in the validation cohort (69% and 92%, respectively) were comparable to those in the learning cohort. The likelihood ratio of the flow cytometric score was 10. Conclusions A flow cytometric score may help to establish the diagnosis of myelodysplastic syndrome, especially when morphology and cytogenetics are indeterminate.

Assessment of minimal residual disease in acute myeloid leukemia.

Purpose of review The last 15 years have witnessed significant improvements in technologies to detect minimal residual disease (MRD) in acute myeloid leukemia (AML). We review recent studies highlighting the potential for novel and standardized assays to provide independent prognostic information and develop more personalized treatment approaches. Recent findings Progress includes establishment of optimized real-time quantitative PCR (RQ-PCR) assays for WT1 (commonly overexpressed and a putative therapeutic target) and mutant NPM1 genes. Moreover, sequential MRD monitoring using an internationally standardized PML-RARA RQ-PCR assay has been successfully used to guide molecularly targeted therapy in acute promyelocytic leukemia (APL). There have also been significant advances in multiparameter flow cytometry (MFC) to detect MRD, with introduction of 6–10 color technology and improved understanding of the immunophenotype of leukemic stem cells. Summary Sensitive MRD detection using MFC and/or RQ-PCR has become feasible in virtually all AML patients. MRD monitoring is now considered a standard of care in APL. Recent studies provide a strong rationale for prospective trials investigating the merits of extending MRD detection to alter therapy and potentially improve outcome in other AML subtypes.

Development of minimal residual disease-directed therapy in acute myeloid leukemia

The last three decades have seen major advances in understanding the genetic basis of acute myeloid leukemia (AML). Comprehensive molecular and cytogenetic analysis can distinguish biologically and prognostically distinct disease subsets that demand differing treatment approaches. Definition of these pretreatment characteristics coupled with morphological response to induction chemotherapy provides the framework for current risk-stratification schemes, aimed at identifying subgroups most (and least) likely to benefit from allogeneic transplant. However, since such parameters lack the precision to distinguish the individual patient likely to be cured with conventional therapy from those destined to relapse, there has been considerable interest in development of multiparameter flow cytometry, identifying leukemia-associated aberrant phenotypes, and real-time quantitative polymerase chain reaction (RQ-PCR) detecting leukemia-specific targets (eg, fusion gene transcripts, NPM1 mutation) or genes overexpressed in AML (eg, WT1), to provide a more precise measure of disease response. Minimal residual disease (MRD) monitoring has been shown to be a powerful independent prognostic factor and is now routinely used to guide therapy in patients with the acute promyelocytic leukemia (APL) subtype. We consider the challenges involved in extending this concept, to develop a more tailored personalized medicine approach to improve the management and outcome of other forms of AML.

MHC class-I associated phosphopeptides are the targets of memory-like immunity in leukemia

Immunity against phosphopeptide antigens lacking in leukemia patients can be restored with stem cell transplantation. Adding to the Toolkit for Cancer Therapy The immune system is increasingly being used as a tool for cancer therapy. Researchers have harnessed the body’s own defense system to specifically target tumors. However, one limitation of immune-targeting strategies is the relative lack of targets. Because cancer cells are derived from normal human tissue, ideal antigens would be specifically or differentially expressed by tumor cells and healthy tissues. Now, Cobbold et al. find that phosphoproteins may broaden the pool of tumor antigens that can be targeted with immunotherapy. One difference between cancer and normal cells is the way in which they are regulated. Indeed, signal transduction pathways are frequently dysregulated in cancer cells. The authors now use a hallmark of signal transduction—protein phosphorylation—to identify and characterize new phosphoantigens that stimulate immune cells. They identified 95 phosphopeptides presented on the surface of leukemic cells and demonstrated that they could be recognized and killed by phosphopeptide-specific cytotoxic T lymphocytes. Somewhat surprisingly, healthy individuals had high levels of responses to phosphopeptides, but these responses were muted in leukemia patients with poor prognosis. What’s more, allogeneic stem cell transplant could restore phosphoprotein immune response in patients. These data suggest that phosphopeptides could be developed as new targets for cancer immunotherapy. Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8+ T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen–matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8+ T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

A Novel Role for PECAM-1 (CD31) in Regulating Haematopoietic Progenitor Cell Compartmentalization between the Peripheral Blood and Bone Marrow

Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1−/− mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1−/− bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the blood