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.

Kinetic characterization of hexokinase isoenzymes from glioma cells: implications for FDG imaging of human brain tumors.

Quantitative imaging of glucose metabolism of human brain tumors with PET utilizes 2-[(18)F]-fluorodeoxy-D-glucose (FDG) and a conversion factor called the lumped constant (LC), which relates the metabolic rate of FDG to glucose. Since tumors have greater uptake of FDG than would be predicted by the metabolism of native glucose, the characteristic of tumors that governs the uptake of FDG must be part of the LC. The LC is chiefly determined by the phosphorylation ratio (PR), which is comprised of the kinetic parameters (Km and Vmax) of hexokinase (HK) for glucose as well as for FDG (LC proportional to (Km(glc) x Vmax(FDG))/(Km(FDG) x Vmax(glc)). The value of the LC has been estimated from imaging studies, but not validated in vitro from HK kinetic parameters. In this study we measured the kinetic constants of bovine and 36B-10 rat glioma HK I (predominant in normal brain) and 36B-10 glioma HK II (increased in brain tumors) for the hexose substrates glucose, 2-deoxy-D-glucose (2DG) and FDG. Our principal results show that the KmGlc < KmFDG << Km2DG and that PR2DG < PRFDG. The FDG LC calculated from our kinetic parameters for normal brain, possessing predominantly HK I, would be higher than the normal brain LC predicted from animal studies using 2DG or human PET studies using FDG or 2DG. These results also suggest that a shift from HK I to HK II, which has been observed to increase in brain tumors, would have little effect on the value of the tumor LC.

Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils.

BACKGROUND: Eosinophils, basophils, and mast cells are believed to be the central tenet cells in allergic conditions including allergic rhinitis, asthma, and eczema. The molecular mechanisms underlying the recruitment of these cells to sites of allergic inflammation are poorly understood. OBJECTIVES: Our aim was to identify a common adhesion molecule that could potentially be responsible for mediating the recruitment of the allergic cell types to the lungs and other sites of allergy. METHODS: We have cloned a sialoadhesin molecule from a human eosinophil library with the use of expressed sequence tag technology and characterized its expression on allergic cells by the use of flow cytometry and specific mAbs. RESULTS: With the use of expressed sequence tag sequencing, we have identified a novel siglec molecule, SAF-2. SAF-2 has homology with other sialoadhesin family members (CD33 and siglec-5) and belongs to a subgroup of the Ig superfamily. SAF-2 is a 431-amino acid protein composed of 3 Ig domains with a 358-amino acid extracellular domain and a 47-amino acid tail. SAF-2 is highly restricted to eosinophils, basophils, and mast cells. Antibodies to SAF-2 do not modulate Ca(++) mobilization or chemotaxis of human eosinophils induced by eotaxin. CONCLUSION: SAF-2 is a highly restricted sialoadhesin molecule, which may be useful in the detection and/or modulation of allergic cells.

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.

Glucose metabolism in human malignant gliomas measured quantitatively with PET, 1-[C-11]glucose and FDG: analysis of the FDG lumped constant.

UNLABELLED: Calculation of the glucose metabolic rate (MRGlc) in brain with PET and 2-[18F]fluoro-2-deoxy-D-glucose (FDG) requires knowing the rate of uptake of FDG relative to glucose from plasma into metabolite pools in the tissue. The proportionality factor for this is the FDG lumped constant (LC[FDG]), the ratio of the volumes of distribution of FDG and glucose multiplied by the hexokinase phosphorylation ratio for the two hexoses, Km(Glc) x Vm(FDG)/Km(FDG) x Vm(Glc) x MRGlc equals the FDG metabolic rate (MRFDG) divided by the LC(FDG), i.e., MRGlc = MRFDG/LC(FDG) and LC(FDG) = MRFDG/MRGlc. This investigation tested the hypothesis that LC(FDG) is significantly higher in gliomas than it is in brain uninvolved with tumor. METHODS: We imaged 40 patients with malignant gliomas with 1-[11C]glucose followed by FDG. The metabolic rates MRGlc and MRFDG were estimated for glioma and contralateral brain regions of interest by an optimization program based on three-compartment, four-rate constant models for the two hexoses. RESULTS: The LC(FDG), estimated as MRFDG/MRGlc, in gliomas was 1.40 +/- 0.46 (mean +/- s.d.; range = 0.72-3.10), whereas in non-tumor-bearing contralateral brain, it was 0.86 +/- 0.14 (range = 0.61-1.21) (p < 0.001, glioma versus contralateral brain). CONCLUSION: These data strongly suggest that the glioma LC(FDG) exceeds that of contralateral brain, that quantitation of the glioma MRGlc with FDG requires knowing the LC(FDG) specific for the glioma and that the LC(FDG) of normal brain is higher than previously reported estimates of about 0.50. 2-Fluoro-2-deoxy-D-glucose/PET studies in which glioma glucose metabolism is calculated by the autoradiographic approach with normal brain rate constants and LC(FDG) will overestimate glioma MRGlc, to the extent that the glioma LC(FDG) exceeds the normal brain LC(FDG). "Hot spots" visualized in FDG/PET studies of gliomas represent regions where MRGlc, LC(FDG) or their product is higher in glioma than it is in uninvolved brain tissue.

Feasibility of imaging pentose cycle glucose metabolism in gliomas with PET: studies in rat brain tumor models.

UNLABELLED: The feasibility of imaging pentose cycle (PC) glucose utilization in human gliomas with PET was explored in two rat glioma models by means of glucose radiolabeled in either the carbon-1 (C-1) or carbon-6 (C-6) position. METHODS: In vitro, monolayers of T-36B-10 glioma, tissue slices of intracerebral glioma grafts or slices of normal brain were fed [1-14C]glucose or [6-14C]glucose, and the generated [14C]CO2 was trapped to quantitate the ratio of [14C]CO2 from 14C-1 versus 14C-6. In vivo, rats bearing grafts of either T-36B-10 or T-C6 rat gliomas at six subcutaneous sites received simultaneous intravenous injections of either [1-11C]glucose and [6-14C]glucose, or [1-14C]glucose and [6-11C]glucose. Tumors were excised between 5 and 55 min postinjection to quantify tracer uptake while arterial plasma was collected to derive time-activity input curves. RESULTS: In vitro, the C-1/C-6 ratio for CO2 production from T-36B-10 monolayers was 8.8 +/- 0.4 (s.d.), in glioma slices it was 6.1 +/- 2.1 and in normal brain slices it was 1.1 +/- 0.7. PC metabolism in T-36B-10 was 1.8% +/- 0.5 of total glucose utilization. In vivo, tumor radioactivity levels normalized by plasma isotopic glucose levels showed that retained C-1 relative to C-6 radiolabeled glucose was significantly lower in both gliomas, 4.9% lower in T-36B-10 (p < 0.01) and 4.7% lower in T-C6 (p < 0.01). In an additional group of rats bearing T-36B-10 gliomas and exposed to 10 Gy of 137Cs irradiation 4 hr before isotope injection, the C-1 level was 5.6% lower than that for C-6 (p < 0.05). These results were analyzed with a model of glucose metabolism that simultaneously optimized parameters for C-1 and C-6 glucose kinetics by simulating the C-1 and C-6 tumor time-activity curves. The rate constant for loss of radiolabeled carbon from the tumors, k4, was higher for C-1 than for C-6 in all groups of rats (19% higher for T-36B-10 unirradiated, 32% for T-36B-10 irradiated and 32% for T-C6 unirradiated). CONCLUSION: Mathematical modeling, Monte Carlo simulations and construction of receiver-operator-characteristic curves show that if human gliomas have a similar fractional use of the PC, it should be measurable with PET using sequential studies with [1-11C]glucose and [6-11C]glucose.

Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules.

CD33 is a member of the Ig superfamily that is restricted to cells of the myelomonocytic lineage but whose functions and binding properties are unknown. It shares sequence similarity with sialoadhesin, CD22, and the myelin-associated glycoprotein, which constitute the Sialoadhesin family of sialic acid-dependent cell adhesion molecules. In the present study, we show that CD33 is a fourth member of this family. As a model for sialic acid-dependent binding, human erythrocytes were derivatized with N-acetylneuraminic acid (NeuAc) in different linkages. A recombinant soluble form of CD33, Fc-CD33, bound red blood cells with a specificity similar to that of sialoadhesin, preferring NeuAc alpha 2,3Gal in N- and O-glycans over NeuAc alpha 2,6Gal in N-glycans. Fc-CD33 also bound selectively to the myeloid cell lines HL-60 and U937. However, CD33 was unable to mediate cell binding after transient expression in COS cells, despite high levels of surface expression. Pretreatment of the CD33-transfected cells with sialidase rendered them capable of mediating sialic acid-dependent binding. These results show that CD33 can function as a sialic acid-dependent cell adhesion molecule and that binding can be modulated by endogenous sialoglycoconjugates when CD33 is expressed in a plasma membrane.