Mark E. Costlow

Clinical relevance of lymphoblast biological features in children with acute lymphoblastic leukemia.

Improvements in therapy for childhood acute lymphoblastic leukemia (ALL) have led us to reevaluate the prognostic significance of lymphoblast characteristics at diagnosis. From application of univariate and multivariate statistical methods, we determined the relationship of five blast cell features to treatment outcome in 250 patients who were enrolled in two clinical trials at this center from May 1979 through April 1982. Karyotype ploidy, lymphoblast morphology, and immunophenotype were each significantly related to prognosis as measured by time to failure, while periodic acid-Schiff reactivity and glucocorticoid receptor number lacked prognostic implication for this patient population. In addition, clinical features of initial WBC count, age, and race were also significant independent variables in predicting treatment response. By multivariate analysis, both ploidy and morphology contributed prognostic information to a clinical model based on WBC count, age, and race. If the model was adjusted for impact of ploidy, however, French-American-British morphology no longer contributed additional prognostic information. Our findings suggest that many traditional biological features used to estimate prognosis in ALL can be discarded in favor of clinical features (leukocyte count, age, and race) and cytogenetics (ploidy) for planning of future clinical trials.

The relationship of blast cell glucocorticoid receptor levels to response to single-agent steroid trial and remission response in children with acute lymphoblastic leukemia☆

Of 263 children with glucocorticoid receptor (GR) levels measured at diagnosis of acute lymphoblastic leukemia (ALL), 27 received single-agent glucocorticoid before combination induction chemotherapy and were evaluable for in vivo clinical response to steroid. Twenty-one were glucocorticoid-responsive and 6 were resistant. There was no difference between the two groups in the distribution of age, sex, white blood cell count, immunophenotype of blasts, initial central nervous system disease or mediastinal mass. The median GR level, however, was appreciably lower in the group of patients with resistant disease (6250 vs 17,800 sites/cell, p = 0.06). Five of 12 patients with GR levels of less than 10,000 sites/cell compared to 1 of 15 with higher levels had glucocorticoid-resistant ALL (p = 0.03). All 21 patients with glucocorticoid-sensitive disease achieved a complete remission after combination induction chemotherapy, but only 3 of 5 evaluable patients in the other group did (p less than 0.04). Two patients were studied both at diagnosis and at relapse; both had decreased GR levels at relapse (below detection in one) and failed to respond to glucocorticoid. We conclude that a lower GR level is associated with glucocorticoid resistance and furthermore that a decrease in the level of GR is a mechanism of acquiring steroid resistance.

Concanavalin a induced alterations in 125I-labeled prolactin binding

Summary 125 I-labeled ovine prolactin binding to receptors in the liver of female rats is markedly inhibited by Concanavalin A (Con A). Lens culinaris agglutinin and succinyl-Con A inhibit binding to a much lesser extent and wheat germ agglutinin and phytohemagglutinin-P are without affect. Con A inhibition is dose related and saturable; however, 30% of prolactin receptors are unaffected by Con A. α-Methylmannoside or α-methylglucoside, both potent inhibitors of Con A binding, reverse the inhibition. Con A also inhibits prolactin binding in other normal target tissues and in neoplastic rat mammary tissue. In contrast, 125 I-labeled Con A binding to rat liver is unaltered by prolactin. These results suggest that Con A binding sites may not be identical to the prolactin receptor and that prolactin receptors may exist in two domains on the cell surface — one of which is in close proximity to Con A binding sites and another which is more distant.

Sequential studies of lymphoblast glucocorticoid receptor levels at diagnosis and relapse in childhood leukemia: An update☆

For 80 children with relapsed acute lymphoblastic leukemia, glucocorticoid receptor (GR) levels were lower in blast cells from patients who developed relapse while receiving chemotherapy (p = 0.03) and in those who failed reinduction treatment (p = 0.07). Serial determinations of GR at diagnosis and at relapse in blast cells from 41 patients disclosed various changes in receptor content, which were not related to the initial GR levels, the immunophenotype of blast cells or number of relapses. Six of 9 patients for whom reinduction therapy failed and only 7 of 32 patients in whom subsequent remission was induced had decreased GR levels at relapse (p = 0.018). One patient failed reinduction despite a sharp increase in GR level. Although a decrease in GR levels between diagnosis and relapse is associated with steroid resistance, other mechanism(s) can also be responsible for the development of steroid resistance in childhood leukemia.

Response of recurrent acute lymphoblastic leukemia to glucocorticoids: Serial studies of receptor content, in vivo cytokinetic changes and clinical responses☆

The sensitivity of relapsed acute lymphoblastic leukemia (ALL) to treatment with steroids was assessed by measuring bone marrow blast, whole-cell glucocorticoid receptor (GR) levels and serial cytokinetic and clinical responses to steroids, both as single agents and in combination reinduction chemotherapy. GR levels ranged widely, from 1800 to 47,800 sites/cell (median 16,000), and did not differ significantly from levels measured in newly diagnosed patients (p = 0.50). Nineteen of the 48 children studied had GR levels measured both at diagnosis and relapse, and in 14 the values at relapse were either decreased or increased by more than 25%. For nine children, the sensitivity to a three-day oral course of dexamethasone alone (6 mg/m2 per day) was estimated from daily leukocyte counts and serial bone marrow cytokinetic studies. A clinical oncolytic effect of dexamethasone was associated with a reduction in the marrow cell proliferating compartment, as judged from labeling indices, mitotic indices and percent S + G2 + M in five patients. We found, however, that GR levels either alone or combined with clinical or cytokinetic responses to dexamethasone as a single agent did not reliably identify patients who were likely to respond favorably to reinduction with steroid-containing combination chemotherapy. The 35 children who successfully attained subsequent remissions had GR levels similar to those who failed (p = 0.35). While not statistically significant, two observations suggest an association between lower receptor content and drug resistance. Receptor levels from patients with ALL who relapsed while on chemotherapy were appreciably lower (median 15,700, n = 31) than GR levels from patients who relapsed off therapy (median 20,300, n = 17) (p = 0.08). Moreover, three of seven patients with serial studies, whose GR levels at relapse were lower than at diagnosis, failed reinduction--compared to only one of twelve whose levels were either increased or remained unchanged (p = 0.11). Although not having any obvious clinical utility, studies of GR at diagnosis and at relapse may aid in clarifying mechanisms of drug resistance in leukemic blasts.

Prolactin receptors in primary cultures of carcinogen-induced rat-mammary tumors.

7,12-Dimethylbenz[alpha]anthracene-induced rat mammary tumors were dissociated with collagenase and hyaluronidase and placed into primary culture. In most cultures, specific binding of 125I-labeled ovine prolactin was (i) lower than that for the original tumors unless bovine prolactin (1 microgram/ml) had been added to the dissociation medium, and (ii) varied with the type of growth medium used. The level of prolactin binding in cultured cells was relatively constant for the first 7-10 days. Prolactin binding in cultured cell homogenates was maximal at pH 7.0, proportional to cell protein, specific for prolactin, and reached a steady state by 12 h at 22 degrees C. The half-maximum inhibition of 125I-labeled prolactin binding by unlabeled prolactin was 100 ng/ml for cells grown in 5-1000 ng of prolactin/ml. After prolactin was removed from the growth medium, the level of available binding sites progressively increased, reached a maximum at 48 h and then declined. At 48 h, the dissociation constant for prolactin binding (Kd approximately 1 x 10(-10) M) was comparable to that in tumors. In some cultured tumors, a 48-h treatment with 0.5 or 1.0 ng of prolactin/ml caused an apparent increase in the level of prolactin binding. Prolactin increased DNA synthesis and its removal caused a reduction in [3H]estradiol and [3H]-R5020 binding to cultured cell cytosols.

Glucocorticoid receptors in childhood acute non-lymphocytic leukemia

Using a whole-cell assay, we found glucocorticoid receptors (GR) in all 43 consecutive assessable children with newly diagnosed acute non-lymphocytic leukemia (ANLL). The receptor levels ranged from 2146 to 81,308 sites/cell (median = 18,105); these results were similar to those for acute lymphocytic leukemia. Receptor levels were not related to any of these clinical or biological features at diagnosis: age, sex, race, initial leukocyte count, liver or spleen size, presence of CNS disease or Auer rods, [3H] thymidine ( [3H]TdR) labelling index, French-American-British morphology or terminal deoxynucleotidyl transferase activity. Receptor levels also were not related to the initial treatment response or remission duration after therapy that did not include a glucocorticoid. We conclude that GR level has no clinical utility as a marker protein in childhood ANLL.

Relationship of prolactin receptors to concanavalin A binding.

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.

Nuclear translocation of lymphoblast glucocorticoid receptors in childhood leukemia does not predict steroid responsiveness

Nuclear translocation of glucocorticoid receptors (GR) was measured in lymphoblasts from 45 children with acute lymphoblastic leukemia (ALL). The percent of total GR translocated to the nucleus ranged from 0 to 100% (mean, 59%) and was independent of the total GR level. Neither the extent of GR nuclear translocation nor the absolute amount of GR translocated at 4 or 25 degrees C correlated with glucocorticoid response in 13 patients with single-agent glucocorticoid trial. These results indicate that defects in nuclear translocation of GR are not a common cause for steroid resistance in childhood ALL.

Metabolic inhibitors increase prolactin binding to cultured mammary tumor cells

Abstract We determined the effects of metabolic inhibitors on 125 I-labeled prolactin binding in monolayers of cultured rat mammary tumors. Chemical agents that blocked energy production increased binding by 8–20 fold, as did lowering the temperature from 37°C to 4°C. This difference was not due to blocking degradation of the hormone and inhibitors of degradation (lysosomotropic amines, bacitracin) did not increase binding. In the presence of a metabolic inhibitor at 37°C, binding reached a steady state within 3 h and had an apparent dissociation constant of ∼6 × 10 −10 M. Studies with fresh tumor slices produced comparable results. The findings indicate that the level of metabolic energy in mammary tumor cells can regulate prolactin binding.