Paul Virgo

Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia

The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

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.

Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting;

Flow cytometry to detect minimal residual disease is a sensitive and specific means to detect impending relapse in B lineage acute lymphoblastic leukemia (ALL). However as with all relatively sophisticated procedures assurance is needed that the methodology is widely applicable, and reproducible in different laboratories. In this paper, the UK ALL Flow MRD Group and the UK MRD steering group describe a four color flow protocol that was applicable to most patients and showed similarly high levels of concordance both between different laboratories and with MRD as detected molecularly. See related perspective article on page 748. Minimal residual disease detection, used for clinical management of children with acute lymphoblastic leukemia, can be performed by molecular analysis of antigen-receptor gene rearrangements or by flow cytometric analysis of aberrant immunophenotypes. For flow minimal residual disease to be incorporated into larger national and international trials, a quality assured, standardized method is needed which can be performed in a multi-center setting. We report a four color, flow cytometric protocol established and validated by the UK acute lymphoblastic leukemia Flow minimal residual disease group. Quality assurance testing gave high inter-laboratory agreement with no values differing from a median consensus value by more than one point on a logarithmic scale. Prospective screening of B-ALL patients (n=206) showed the method was applicable to 88.3% of patients. The minimal residual disease in bone marrow aspirates was quantified and compared to molecular data. The combined risk category concordance (minimal residual disease levels above or below 0.01%) was 86% (n=134). Thus, this standardized protocol is highly reproducible between laboratories, sensitive, applicable, and shows good concordance with molecular-based analysis.

Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage.

Quek and colleagues identify human leukemic stem cells (LSCs) present in CD34− AML. In-depth characterization of the functional and clonal aspects of CD34− LSCs indicates that most are similar to myeloid precursors.

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.

Identification of the CD33-related siglec receptor, Siglec-5 (CD170), as a useful marker in both normal myelopoiesis and acute myeloid leukaemias

Summary. Sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐5 or CD170 is a CD33‐related receptor, containing cytoplasmic immune receptor‐based tyrosine signalling motifs, that has previously been reported to be myeloid‐specific like CD33 and thus may be useful in the characterization of both normal and malignant haemopoiesis. This study showed that Siglec‐5 had a distinct expression pattern to CD33 both on normal myeloid cells and in acute myeloid leukaemia (AML). In normal bone marrow and cord blood, myeloid cells predominantly expressed Siglec‐5 at the later stages of granulocytic differentiation. Siglec‐5 was not expressed at significant levels by CD34+ progenitors either from bone marrow or mobilized peripheral blood. During in vitro myeloid differentiation of cord blood purified CD34+ cells, Siglec‐5 was upregulated later than CD33. Siglec‐5 expression remained absent or very low on cultured CD34+ cells, unlike CD33, which was present on almost all CD34+ cells by day 4. However, analysis of blasts from 23 patients with AML revealed aberrant expression of Siglec‐5 with CD34 in 50% (seven of 14) of patients with CD34+ AML; 61% (14 of 23) of AML cases were positive for Siglec‐5 with an increased frequency in the French–American–British subtypes M3‐5 (80%) compared with M0‐2 (25%). All 13 acute lymphoblastic leukaemic (ALL) samples tested, including a CD33+ ALL, were Siglec‐5 negative. These results support the further evaluation of Siglec‐5 antibodies in the diagnosis and monitoring of AML.

A prospective trial evaluating the role of mesothelin in undiagnosed pleural effusions

Mesothelin has been proposed as a useful tool in the diagnosis of malignant pleural mesothelioma (MPM). We aimed to examine its diagnostic utility and the impact of renal impairment on results. We prospectively recruited 230 patients with new undiagnosed pleural effusions, testing serum (n=216) and pleural fluid (n=206) mesothelin (by ELISA) during the initial consultation. 28 (12%) out of 230 patients had MPM. Serum mesothelin gave sensitivity 59.3%, specificity 64.7%, negative predictive value (NPV) 91.2%, positive predictive value (PPV) 20.5%, and pleural fluid sensitivity 72.0%, specificity 87.5%, NPV 95.5%, PPV 46.2% for distinguishing effusions due to MPM. In a matched comparison, diagnostic characteristics of pleural fluid mesothelin were superior to serum (p=0.0001). Serum mesothelin levels in patients without MPM were higher in patients with renal impairment (p=0.007) while pleural fluid levels were unaffected. 19 (54%) out of 35 patients with a benign pleural effusion and an estimated glomerular filtration rate ≤59 mL·min−1 had a false-positive serum mesothelin result. The diagnostic accuracy of pleural fluid mesothelin is superior to that of serum and is unaffected by renal function. In patients with a low pre-test probability of mesothelioma, a negative mesothelin test could be reassuring, because of its high NPV. Routine use of mesothelin testing in undiagnosed pleural effusions at presentation appears to be unhelpful.

Flow cytometry in clinical pathology

Flow cytometry has had an impact upon all areas of clinical pathology and now, in the 21st century, it is truly coming of age. This study reviews the application of flow cytometry within clinical pathology with an emphasis upon haematology and immunology. The basic principles of flow cytometry are discussed, including the principles and considerations of the flow-cell and hydrodynamic focusing, detector layout and function, use of fluorochromes and multicolour flow cytometry (spectral overlap and colour compensation), alongside the strategies available for sample preparation, data acquisition and analysis, reporting of results, internal quality control, external quality assessment and flow sorting. The practice of flow cytometry is discussed, including the principles and pitfalls associated with leukocyte immunophenotyping for leukaemia and lymphoma diagnosis, immune deficiency, predicting and monitoring response to monoclonal antibody therapy, rare event detection and screening for genetic disease. Each section is illustrated with a case study. Future directions are also discussed.

Expression of CD33 is a predictive factor for effect of gemtuzumab ozogamicin at different doses in adult acute myeloid leukaemia.

It remains unclear in adult acute myeloid leukaemia (AML) whether leukaemic expression of CD33, the target antigen for gemtuzumab ozogamicin (GO), adds prognostic information on GO effectiveness at different doses. CD33 expression quantified in 1583 patients recruited to UK-NCRI-AML17 (younger adults) and UK-NCRI-AML16 (older adults) trials was correlated with clinical outcomes and benefit from GO including a dose randomisation. CD33 expression associated with genetic subgroups, including lower levels in both adverse karyotype and core-binding factor (CBF)-AML, but was not independently prognostic. When comparing GO versus no GO (n=393, CBF-AMLs excluded) by stratified subgroup-adjusted analysis, patients with lowest quartile (Q1) %CD33-positivity had no benefit from GO (relapse risk, HR 2.41 (1.27–4.56), P=0.009 for trend; overall survival, HR 1.52 (0.92–2.52)). However, from the dose randomisation (NCRI-AML17, n=464, CBF-AMLs included), 6 mg/m2 GO only had a relapse benefit without increased early mortality in CD33-low (Q1) patients (relapse risk HR 0.64 (0.36–1.12) versus 1.70 (0.99–2.92) for CD33-high, P=0.007 for trend). Thus CD33 expression is a predictive factor for GO effect in adult AML; although GO does not appear to benefit the non-CBF AML patients with lowest CD33 expression a higher GO dose may be more effective for CD33-low but not CD33-high younger adults.

Immunoglobulin E deficiency: a forgotten clue pointing to possible immunodeficiency?

Background Patients with primary antibody deficiency often have delayed diagnosis. Very low IgE, found during investigations for allergy, may be a marker for other immunodeficiency. Methods We introduced a new laboratory policy of testing cases with very low IgE levels for possible linked antibody deficiency. The data represent an audit of routine results collected over two years. Results Very low IgE (≤2 IU/mL) was identified in 85/2622 (3.2%) routine patient samples. Two children and four adult patients were found to have one or more classes of immunoglobulin below the reference range for age. In 2/6, the initiative of the laboratory led to a new unsuspected diagnosis of antibody immunodeficiency. Conclusions Common variable immunodeficiency continues to be overlooked as a primary cause of lung disease in adults. Very low serum IgE should trigger appropriate investigation (immunoglobulin quantification and serum electrophoresis).