Threshold segmentation for PET target volume delineation in radiation treatment planning: the role of target-to-background ratio and target size.

A multivariable approach was adopted to study the dependence of the percentage threshold [TH(%)] used to define the boundaries of 18F-FDG positive tissue on emission scan duration (ESD) and activity at the start of acquisition (Aacq) for different target sizes and target-to-background (T/B) ratios. An anthropomorphic model, at least for counting rate characteristics, was used to study this dependence in conditions resembling the ones that can be encountered in the clinical studies. An annular ring of water bags of 3 cm thickness was fitted over an International Electro-technical Commission (IEC) phantom in order to obtain counting rates similar to those found in average patients. The scatter fraction of the modified IEC phantom was similar to the mean scatter fraction measured on patients, with a similar scanner. A supplemental set of microhollow spheres was positioned inside the phantom. The NEMA NU 2-2001 scatter phantom was positioned at the end of the IEC phantom to approximate the clinical situation of having activity that extends beyond the scanner field of view. The phantoms were filled with a solution of water and 18F (12 kBq/mL) and the spheres with various T/B ratios of 22.5, 10.3, and 3.6. Sequential imaging was performed to acquire PET images with varying background activity concentrations of about 12, 9, 6.4, 5.3, and 3.1 kBq/mL. The ESD was set to 60, 120, 180, and 240 s/bed. Data were fitted using two distinct multiple linear regression models for sphere ID < or = 10 mm and sphere ID > 10 mm. The fittings of both models were good with an R2 of 0.86 in both cases. Neither ESD nor Aacq resulted as significant predictors of the TH(%). For sphere ID < or =10 mm the target size was the most significant predictor of the TH(%), followed by the T/B ratio, while for sphere ID > 10 mm the explanatory power of the target size and T/B ratio were reversed, the T/B ratio being now the most important predictor of the TH(%). Both the target size and T/B ratio play a major role in explaining the variance of the TH(%), throughout the whole range of target sizes and T/B ratios examined. Thus, algorithms aimed at automatic threshold segmentation should incorporate both variables with a relative weight which critically depends on target size.

20th-century advances in drug therapy in oncology--Part. II.

Ongoing research in cancer therapy has led to the development of antineoplastic agents which target specific components of the cell cycle. In Part II of this series, we discuss agents which target the mitotic mechanism by inhibiting microtubules. Although many of these agents are being shown to have multiple effects, the Vinca alkaloids and the taxanes are known as antimitotic drugs. They are among the most important anticancer agents currently available, and because of their unique mechanisms, can be combined with a wide variety of other antineoplastic agents in a spectrum of diseases. In addition, in part II, we are discussing agents that target DNA and prevent replication and thus cell growth by inhibiting the enzymes which protect DNA during replication, the topoisomerases. These drugs, too, have unique mechanisms of action and have become major components of combination regimens. The topoisomerase I inhibitors are new drugs derived from an older parent drug, and their full possibilities are still being explored.

Acute leukemia with t(10;11)(p11-p15;q13-q23).

We report six patients with acute leukemia characterized by the presence of a t(10;11)(p11-15;q13-q23), either as sole cytogenetic abnormality (three patients) or as part of a complex abnormal karyotype. The morphologic and cytochemical features of four patients were consistent with FAB-M5A, while two patients presented with FAB-L1 characteristics. By immunophenotyping, myeloid leukemia was diagnosed in five patients, including one patient with FAB-L1 leukemia who typed as terminal transferase (TdT)+, CD7 T-cell antigen+ acute myelomonocytic leukemia. Differentiated acute myeloid leukemia (AML) with expression of terminal transferase was found in two of the other cases and monocytic leukemia in two, with co-expression of T-cell antigens in one of them. The second FAB-L1 patient typed as undifferentiated acute lymphocytic leukemia (ALL) expressing myeloid antigens. Serial phenotypic studies in patient 3 during the course of the disease demonstrated a switch from monocytic to lymphoid morphology at the time of first and second relapse, which was paralleled by the appearance of a pre-T ALL immunophenotype with co-expression of the myeloid antigen CD33. These phenotypic changes occurred without apparent alteration in the genotype since t(10;11)(p11.2;q23) remained the only cytogenetic aberration at all stages of the disease. Our observations suggest that the (10;11) variant of 11q aberrations occurs in a bipotential myelomonocytic/T-lymphoid stem cell.

The binding activity of estrogen receptor to DNA and heat shock protein (Mr 90,000) is dependent on receptor-bound metal.

1,10-Phenanthroline inhibited the DNA-cellulose binding of the transformed calf uterus estrogen receptor (homodimer of 66-kDa molecules: 5 S estrogen receptor) in a temperature- and concentration-dependent manner. This result appears related to the metal-chelating property of 1,10-phenanthroline, since the inhibition was decreased by addition of Zn2+ and Cd2+, but not by Ca2+, Ba2+, or Mg2+ for which the affinity of the chelator is low. Only a slight inhibition was observed in the presence of the 1,7-phenanthroline, a nonchelating analogue. After dialysis or filtration to remove free 1,10-phenanthroline, DNA binding of the 5 S estrogen receptor was still inhibited. Conversely, the chelator was unable to release prebound 5 S estrogen receptor from DNA-cellulose. The 5 S estrogen receptor DNA binding was inhibited when 1,10-phenanthroline was present during the transformation to activated receptor of the hetero-oligomeric nontransformed 9 S estrogen receptor, in which the hormone binding subunits are associated with heat shock protein, Mr 90,000 (hsp 90) molecules. In contrast, if 1,10-phenanthroline was removed before the transformation took place, only a slight inhibition was observed. Other experiments with EDTA indicated a similar inhibition of DNA-cellulose binding by the 5 S estradiol receptor, and all metal ions chelated by this agent prevented its inhibitory effect. The results indicate that 1,10-phenanthroline inhibited the DNA binding of the transformed 5 S estradiol receptor by chelating metal ion tightly bound to the receptor, which is not accessible to the chelator when the receptor is bound to DNA or to hsp 90. Therefore, they suggest that the metal ion may play a critical role in the interaction with DNA and hsp 90 by maintaining the structural integrity of the implicated receptor domain.

Subunit composition of the molybdate-stabilized "8-9 S" nontransformed estradiol receptor purified from calf uterus.

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits.

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography.

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.

Properties of biospecific adsorbents, obtained by immobilization of oestradiol 7 alpha-derivatives, for purification of calf-uterine cytosol oestradiol receptor.

The properties of three types of adsorbents obtained by coupling oestradiol 7 alpha-derivatives to agarose were compared. The adsorbents examined were: oestradiol 7 alpha-decamethylene-agarose, oestradiol 7 alpha-decamethylene-poly(anayl-lysine)-agarose and oestradiol 7 alpha-trimethylene-poly(alanyl-lysine)-agarose. The following results were obtained. (1) All these adsorbents are stable at 0 degrees C for a least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximala receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin-Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is possible only with biospecific adsorbents containing low concentrations of coupled steroid (less than or equal to 0.2 mg/ml). The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr 60000). When starting from a low salt preparation containing the native 8-S receptor, partially purified by heparin-Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250--500-fold and led to a 50--90% pure protein (assuming one oestradiol binding site per molecule of Mr 70000).

[Two-step purification of estradiol calf uterine cytosol receptor]. / Purification en deux étapes du récepteur cytosoluble de l'oestradiol d'utérus de veau.

The use of two affinity chromatography methods: heparin-Ultrogel and estradiol 7 alpha-(CH2)3-poly (alanine-lysine)-agarose, allows us to purify the calf uterine estradiol cytosol receptor with a 20% recovery. Electrophoresis under denaturing conditions allows us to estimate its molecular weight as 70 000 dalton.

[Production and detection of antibody against estradiol receptor in the calf uterus. Interaction with the estrogen receptor from the hen oviduct]. / Production et détection d'anticorps antirécepteur de l'oesstradiol d'utérus de veau. Interaction avec le récepteur d'oviducte de poule.

The antibodies against estrogen receptor were obtained after injecting Rabbits with a cytoplasmic receptor fraction isolated from Calf uterus. The estrogen receptor was partially proteolysed by the action of trypsin and subsequently purified by affinity chromatography (purification 4,000 to 10,000 fold, to a purity of 5-20%). The affinity of the antibody for the proteolysed receptor is KD approximately 1 nM and serum titres have reached values of approximately 50 nM. The values remained constant after the third injection. Preliminary results indicate that the antibody has approximately the same affinity for "native" cytoplasmic estrogen receptor from Calf uterus, as well as for the "trypsinized" forms of estrogen receptor isolated from Calf uterine cytosol and Hen oviduct nuclei.