[CANCER RESEARCH 38, 4251-4256. November 1978]

Clinical Implications of Glucocorticoid Receptors in Human Leukemia1

Marc E. Lippman,2 GéraldineKonior Yarbro,3 and Brigid G. Leventhal

Medicine Branch, National Cancer Institute, Bethesda, Maryland 20014, and [M. E. L ¡,Pediatrie Oncology Division, Johns Hopkins Hospital, Baltimore,Maryland 21202 ¡G K. Y.. B. G. L.¡

Abstract

Glucocorticoid receptors were studied in various populations of normal human peripheral blood lymphocytesand leukemic lymphoblasts. Normal lymphocytes containlow levels of glucocorticoid receptor (-2,500 sites/cell)which are identical in T- and non-T-fractions. Phytohe-magglutinin treatment increases levels about 3-fold. Leukemic lymphoblasts contain larger numbers of receptorsites. Presence of receptor is correlated with in vitrosensitivity to glucocorticoids and in vivo response totherapy. Quantity of receptor is also correlated with complete remission duration independently of leukemic celltype (T or null), initial WBC, or age of patient. Quantitativedetermination of glucocorticoid receptor levels in acutelymphoblastic leukemia may be of value both as anindependent prognostic variable and in suggesting whichpatients should receive glucocorticoid therapy.

Introduction

The inhibitory effects of glucocorticoids on lymphoidtissues have been long appreciated (25, 29). Naturally,therefore, these steroids were widely used in the management of acute leukemia when it became apparent that theycould also kill leukemic lymphoblasts (6, 10). Despite thisuseful observation there are certain difficulties in theireffective use. First, variable response rates are observed inpatients with differing histological diagnoses (22). Secondly, many patients whose disease is initially responsiveto glucocorticoids eventually relapse at a later point in theirclinical course with steroid-resistant cells (38). Thus, whileinitial response rates in ALL" range between 45 and 65%,after relapse the rate of remission induction with glucocorticoids alone falls to 25%. Furthermore, steroid administration is associated with a host of iatrogenic complications,including immunosuppression with concomitant nosocomial infections, Cushing's syndrome, diabetes mellitus, poor

wound healing, peptic ulcer disease, psychosis, and otherproblems (1, 13). These untoward reactions become particularly vexatious when it is recalled that most leukemic andrelated disorders are managed by combinations of cytotoxictherapy which include glucocorticoids along with severalother agents. Thus, possibly harmful components of a drugcombination may be continued long after they have ceasedto be useful. Quite obviously, it would be of value to predict

1 Presented at the John E. Fogarty International Center Conference on

Hormones and Cancer, March 29 to 31,1978. Bethesda, Md.2 To whom requests for reprints should be addressed, at Medical Breast

Cancer Section, Building 10. Room 6B02, NIH, Bethesda, Md. 20014.3 Deceased.' The abbreviation used is: ALL, acute lymphoblastic leukemia.

in advance which leukemic cells might be inhibited byglucocorticoids and thus restrict their use to beneficialsituations. Clearly, an in vitro test of glucocorticoid leukemic cell cytotoxicity would seem worthwhile, but thus farsuch methods have not been widely adopted (7, 8). It isdifficult to reliably culture leukemic cell populations and,furthermore, in vitro effects of hormones may not be aseasily demonstrated as in vivo responses.

Studies performed in breast cancer supported the notionthat quantification of specific steroid receptors for estrogenwas useful in predicting response to endocrine therapy(23). Based upon this fact, plus a clearer understanding ofthe mechanism of action of glucocorticoids (26. 35), itappeared worthwhile to examine human leukemic cells forthe presence of glucocorticoid receptors and to seewhether their presence was correlated with glucocorticoidresponsiveness. This study briefly summarizes our priorwork in characterizing the glucocorticoid receptors of human leukemic cells and outlines some of our more recentdata correlating steroid receptors with clinical features ofthe patients' illness. Much of our earlier work has recently

been reviewed (16), and for this reason most of our effortwill be aimed at presenting more recent studies.

Glucocorticoid Receptors in ALL

Because of the high initial response rate of ALL to glucocorticoids and general patient availability, we initially choseto examine cells from these patients for glucocorticoid receptors (19-21 ). With the use of a competitive protein binding assay on cytoplasmic extracts prepared from eitherviably frozen or fresh lymphoblasts, it was possible to demonstrate saturable binding of radiolabeled glucocorticoid.All samples from patients with untreated ALL containedsaturable receptor sites which ranged in concentration between about 0.12 and 0.8 pmol [3H]dexamethasone boundper mg of cytoplasmic protein. We estimated about 1 x 10~8

mg of cytoplasmic protein per cell (based on cell count andLowry protein determination). This would correspond to between about 1,000 and 8,000 sites/cell. The binding dataobtained, when plotted with the use of the Scatchard technique, were best fitted by a straight line, suggesting thatbinding was to a class of receptor sites of uniform affinity.The equilibrium dissociation constant (Kd) of this receptorranged between 2 and 8 nM in various experiments when[3H]dexamethasone was used as the ligand and was increased about 10-fold when [3H]cortisol was employed. Anequilibrium dissociation constant not significantly differentfrom those values was obtained from the quotient of themeasured association and dissociation rate constants (19).This suggested that true equilibrium conditions were metwith the use of our assay methodology. This binding component was sensitive to proteolytic digestion with trypsin,

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M. E. Lippman et al.

chymotrypsin, or papain, but resistant to RNase, DNase,and phospholipase A, C, or D treatments, suggesting thatthe receptor was a protein. Mercuric chloride preincubationobliterated specific binding while sulfhydryl reagents andglycerol up to 20% concentration stabilized binding activity.

While high-affinity binding is certainly an important criterion for a steroid receptor, it alone is insufficient to establish with certainty that the binding component is functioning as a receptor. For example, plasma transport proteinssuch as transcortin or corticosteroid-binding globulin canalso bind natural glucocorticoid with high affinity. For thisreason the specificity of binding was examined in detail(19). All biologically active glucocorticoids tested competedwith either [3H]cortisol or [3H]dexamethasone for binding.

Unlabeled triamcinolone and dexamethasone, fluorinatedsynthetic glucocorticoids with very low affinity for corticosteroid-binding globulin, showed a higher apparent affinityfor the receptor than did cortisol itself. This parallels theirknown increased biological potency. The receptor showedlittle apparent affinity for compounds without glucocorticoid activity such as metabolites (tetrahydrocortisol) orinactive stereoisomers (11«-hydroxycortisol). Both progesterone and cortexolone, which are inactive as glucocorticoids but act as antiinducers in rodent lymphoid cells (26),were effective competitors for binding. In addition, thecytosolic binding component readily distinguished betweencortisone and cortisol or prednisone and prednisolone,showing that reduction of the 11-keto group (which isnecessary for biological activity) is necessary for optimalbinding. Thus, there are close correlations between extentof binding and biological effect.

Using this cytoplasmic assay we examined the leukemicblast cells of 22 patients for cytosolic glucocorticoid receptor. All patients had some detectable receptor (minimum0.12 pmol [3H]dexamethasone bound per mg of cytoplasmic

protein). Average binding in these untreated patients was0.31 ±0.1 (S.D.) pmol per mg cytosol protein. All of thesepatients subsequently responded to an initial attempt atinduction chemotherapy with a drug combination includingglucocorticoid. Six other patients were studied at the timeof leukemic relapse. At this time, leukemic cells werestudied prior to a reinduction attempt with a drug combination including a glucocorticoid. These 6 patients subsequently achieved a complete remission on this therapy.Their average glucocorticoid binding activity was 0.30 ±0.12 pmol of [3H]dexamethasone per mg of cytoplasmicprotein, a value not significantly different from that inpreviously untreated patients. Six additional patients werestudied at the time of relapse who subsequently failed toachieve a remission on an identical drug combinationwhich included glucocorticoid. Glucocorticoid binding activity in these patients was 0.015 ± 0.0095 pmol[3H]dexamethasone bound per mg of cytoplasmic protein.One patient was studied on 3 separate occasions (his initialpresentation, first relapse, and second relapse). Inductiontherapy was attempted with the identical drug combination,including prednisolone in each case. At presentation, binding activity was 0.32, at first relapse 0.40, and at secondrelapse 0 pmol [3H]dexamethasone bound per mg of cyto

plasmic protein. Induction was successful initially and atthe time of first relapse, but the patient showed no benefi

cial response to therapy on the second attempt at reinduc-tion therapy. With the exception of this particular patient,one cannot conclude that the intervening therapy hadselected out a subpopulation of receptor deficient cells. Aswill be shown later, it is probable that in some cases relapsemay occur in association with préexistent low receptorlevels. Thus, there appeared to be a good correlationbetween response to therapy and the presence of glucocorticoid receptor. This is a somewhat surprising result giventhe fact that these patients were not managed with singleagent therapy, but received drug combinations includingagents which have no apparent biochemical cross-resistance with glucocorticoids (vincristine, methotrexate, and 6-mercaptopurine). Thus, resistance to glucocorticoidswould not necessarily be correlated with either resistanceto other components of the drug combination employed orfailure to observe a clinical response.

In order to learn the biological significance of thesereceptor levels we sought to quantitate the inhibitory effectsof glucocorticoids on human leukemic blast cells with theuse of a direct in vitro assay. Since inhibition of nucleosideincorporation had been shown by other investigators to bean early effect of glucocorticoids on responsive lymphoidcells (28, 34), we measured [3H]thymidine incorporation inshort-term cultures of human leukemic lymphoblasts in thepresence or absence of varying concentrations of glucocorticoid. Unfortunately, these experiments could not be accomplished in every patient. However, in patients whosecells contained binding activity there was a close correlation between concentrations of glucocorticoid which inhibited nucleoside incorporation and concentrations whichbound to receptor sites (19, 20). Furthermore, we neverobserved inhibition of nucleoside incorporation by pharmacologically relevant concentrations of glucocorticoids inleukemic blast cells from patients which lacked bindingactivity. Surprisingly, we never failed to observe inhibitionwhen cytoplasmic binding was present. This was an unanticipated result, given the many steps in steroid hormoneaction distal to the initial binding reaction (35) whichpotentially might be interfered with and yet leave behindcytoplasmic binding activity. We have subsequently described a human ALL cell line in tissue culture in whichalthough there is cytoplasmic binding activity, there are nodemonstrable biological effects of glucocorticoids onamino acid pools, glucose uptake, macromolecular synthesis, or growth (21). Subsequently, various mouse lymphomavariants or mutants derived from a steroid-sensitive mouselymphoma line (S49) have been extensively characterizedby several workers (2, 31, 32, 39). Most of the glucocorti-coid-resistant clones were defective in binding activity, andin all of the remaining clones a receptor defect could alsobe invoked as a potential explanation for hormone unre-sponsiveness. Whatever the explanation for the commonness of receptor defects in glucocorticoid-resistant leukemic cells, it is nonetheless quite interesting that in ourown work there is such a close linkage between receptoractivity and clinical responsiveness, not only to glucocorticoids in vitro but also drug to combinations in general. Thenotion that glucocorticoid binding activity might be important in predicting response to other cytotoxic agents issuggested by these data and will receive further substantia-

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Glucocorticoid Receptors and Leukemia

tion in the data presented below.

Glucocorticoid Receptors in Normal Human Lymphocytes

First, however, we would like to summarize a few of ourstudies which characterize the glucocorticoid receptors ofnormal human lymphocytes (18, 27). We had originallynoted (19) that lymphocytes collected from normal humanvolunteers appeared to have very low levels of glucocorticoid receptor when measured by a cytoplasmic assay (0.03±0.025 pmol [3H]dexamethasone per mg cytoplasmic protein). By determining cytoplasmic protein content on a per-cell basis, this would be equivalent to about 1500 glucocorticoid binding sites/cell. For a variety of reasons it seemedreasonable to repeat and extend these studies to purifiedpopulations and subpopulations of human lymphocytes. Todo this, we switched to a whole cell assay methodologybecause we felt that this assay would be more rapid, moresensitive, and might allow detection of binding sites whichwere noncytoplasmic. This assay has been extensively validated (15). With the use of such a methodology, glucocorticoid receptor activity is readily demonstrable in normalhuman lymphocytes essentially free of polymorphonuclearleukocyte and other mononuclear cell contamination(greater than 99.5% small lymphocytes). Homo and colleagues (11), using a similar assay, have also been able todemonstrate glucocorticoid receptors in normal humanlymphocytes. They used a Ficoll-Hypaque technique (4) toseparate mononuclear cells; however, such preparationscontain significant amounts (up to 10%) of monocytes. Asdescribed later, we have shown that these monocytescontain more than 7000 glucocorticoid receptor sites/cell(18). We found that small peripheral blood lymphocytescontain about 2300 glucocorticoid sites/cell which, in termsof affinity for [3H]dexamethasone (K„,2 to 4 nw) and

specificity studies, are perfectly typical of other glucocorticoid receptors. When these cells are stimulated with themitogen phytohemagglutinin, there is about a 3-fold increase in glucocorticoid receptor activity on a per-cell basis(27). Induction occurs after about a 10- to 12-hr lag. Thisinduction of glucocorticoid binding activity can be totallyinhibited by concurrent incubation with either actinomycinD or cycloheximide. These studies have been confirmed byothers (33). While both "resting" and lectin-stimulated

lymphocytes can be inhibited by glucocorticoids, the degree of inhibition is far higher in the stimulated cells;however, when measured relatively crudely, the sensitivityto hormone is unaltered. However, one might predict thatwhen a given biological effect depends upon the quantityof hormone receptor complex which is formed, rather thanfor any given ambient hormone concentration, the cell withthe highest receptor concentration would have the highestconcentration of hormone receptor complexes, other factors being equal. Thus cells with higher receptor concentrations might be differentially more sensitive to glucocorticoids. Elsewhere in this symposium, a report from thelaboratory of Bourgeois et al. (3) elegantly confirms thishypothesis. These increases in receptor have been suggested to be due to variations in receptor content withdifferent phases of the cell cycle. Cidlowski and Michaels(5) have suggested that in synchronized HeLa cells in

culture there is a doubling of the receptor content on a per-cell basis as the cells pass through late G,. In our studies,phytohemagglutinin stimulates an increase in glucocorticoid receptor activity occurring between 14 and 20 hr,which is well in advance of the entrance of these cells intothe DNA-synthetic phase of the cell cycle (36). These purified lymphocytes consist of about 65 to 75% T-cells and 20to 30% B-cells.

We have recently attempted to further characterize sub-populations of normal human lymphocytes for 2 reasons(first, because of the known differential effects of glucocorticoids on different limbs of the immune system (9) and,secondly, as an attempt to explain differences in receptorcontent in various subsets of human leukemic blast cells).

With the use of rosetting techniques, highly purifiedpopulations of T-, non-T-, and mononuclear cells wereobtained from human peripheral blood, and glucocorticoidbinding activity was determined (18). Results are shown inTable 1. There is no difference in either T- or non-T-receptorcontent on a per-cell basis or in affinity for glucocorticoid.Specificity of the binding site for various steroids was verysimilar as well. Mononuclear cells had about 7,000 sites/cell. Nonstimulated T- and non-T-populations of humanlymphocytes showed an approximately equal degree ofinhibition by glucocorticoids when overall precursor incorporation into macromolecules was measured. Interferencewith specific immune function such as performance incytotoxicity assays or immunoglobulin production was notmeasured. Our data suggest that the differential effects ofglucocorticoids on the immune system do not appear to bemediated by differences in glucocorticoid receptor contentin these cells.

Glucocorticoid Receptors in Subsets of Human ALL

Recently, we have reexamined glucocorticoid receptorsin ALL and attempted to correlate glucocorticoid receptorlevels with different cell subpopulations (14, 15). Approximately one-fifth of patients with ALL have lymphoblastswhich will form spontaneous rosettes with sheep erythro-cytes and are therefore termed T-derived lymphoblasts.These patients will have a disease which frequently presentsdifferently (e.g., mediastinal masses) and which is moreaggressive in that there are very few long-term survivors(12, 30, 37). The majority of patients have lymphoblastswhich lack identifiable cell surface markers (termed nullcells). We have previously shown that lymphoblasts thatmake E-rosettes usually share with normal human E-ro-sette-forming cells the inability to stimulate allogeneic re-sponders in the mixed-lymphocyte culture. We have postulated that the inability or loss of ability to stimulate inmixed-lymphocyte culture may represent maturation along

Table 1Glucocorticoid receptors in subpopulations of human leukocytes

No. of Glucocorticoid Dissocia-experi- receptor levels tion con-ments (sites/cell) slant (nw)

T-lymphocytes 5Non-T-lymphocytes 4Monocytes 2

3130 ±1316 3.5 ±1.42892 ±1068 4.0 ±1.77009 ±1455 5.1

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M. E. L/ppman et al.

the pathway of T-cell differentiation. We therefore subdividenull lymphoblasts into 2 categories, according to whetheror not they stimulate in mixed-lymphocyte culture, for thepurpose of analysis in this paper. We have comparedglucocorticoid receptor levels in these subtypes of ALL andhave determined their relationship to other prognostic variables in these patients. Glucocorticoid receptors are readilydetectable in T- and null leukemic cells. By either affinitycriteria (K,, ~ 4 nM for [3H]dexamethasone) or bindingspecificity studies, T- and null cell leukemic cells are indistinguishable. However, there are significant differences inreceptor content between these cells. Null cells from 18patients which stimulate in mixed-lymphocyte culture (N+ )have a median receptor content of 7,571 sites/cell, a meanof 10,117, and a range of 4,096 to 21,869 sites/cell. Ninepatients had null cells which did not stimulate in mixed-lymphocyte culture (N-). Cells of these patients had amedian of 4,484 sites/cell, with a mean of 6,729 and a rangeof 2,936 to 16,469. Nine patients whose T-lymphoblastswere studied had a median of 2,173 sites/cell, a mean of2,538 and a range of 0 to 5,887. T-lymphoblasts differsignificantly by 2-tailed test from N+ lymphoblasts (p <0.001), as well as N- lymphoblasts (p < 0.01).

Some important patient characteristics as a function ofreceptor content are shown in Table 2. Cells from a total of45 patients eventually were examined for cell surfacemarkers and glucocorticoid receptor content, and thesedata were collated with clinical information from thesepatients. Eleven patients with low levels of receptor (lessthan 2,500 sites/cell) all had T-lymphoblasts. Seven patientswith T- and 11 with null lymphoblasts had intermediatereceptor levels of greater than 2,500, but less than 6.000,sites/cell. Sixteen patients with high receptor levels (greaterthan 6,000 sites/cell) had null lymphoblasts. Patients withT-cell leukemia tend to be older, as has been previouslyshown. Patients with T-cell ALL with lower receptor levelshad higher WBC than did patients with the same cell typewho had higher receptor levels.

We next examined the effect of receptor level on initialcomplete remission induction and duration. Chart 1 showsthe results for patients with T-lymphoblasts. Eleven patientshad less than 2,500 receptor sites/cell, and 7 patients hadbetween 2,500 and 6,000 sites/cell. Two patients in eachgroup failed to achieve a complete remission. While curvesfor remission duration are not quite significantly different(0.05 < p < 0.1) when only patients achieving completeremission are considered, the 2 groups are significantlydifferent with respect to complete remission duration. All11 patients with low receptor levels have relapsed, while 3to 7 patients with intermediate receptor levels remain inremission.

Chart 2 provides a similar analysis of complete remissionduration for patients with null-cell disease. As shown, null-cell patients with high levels of receptor do significantlybetter (p < 0.02) than patients with intermediate receptorvalues.

If Charts 1 and 2 are compared, it can be seen that thecurves for either T- or null-cell patients with intermediatelevels of glucocorticoid receptor are virtually superimposable.

Independent of cell type, glucocorticoid receptor levelwas strongly correlated with remission duration. Resultsare shown in Chart 3. Eleven patients with receptor levelsof 0 to 2,500 sites/cell had a median complete remissionduration of 7.6 months. The relative relapse rate whencompared to the combined groups is 2.76, and all 11patients have relapsed. Eighteen patients with intermediatereceptor levels (7 T and 11 null) had a median remissionduration of 21.4 months. The relative relapse rate of thesepatients is 1.1. Sixteen patients (all null-cell type) with highreceptor levels have a median remission duration in excessof 30.4 months. The relative relapse rate is 0.42. Each of

REMISSION DURATION

Chart 1. Complete remission duration in patients with T-cell-type ALL A,patients with receptor levels ^2500 glucocorticoid sites/cell; •¿�,patientswith receptor levels between 2500 and 6000 glucocorticoid sites/cell; x,patients still in remission

._

•¿�ri•¿�I »j- \

Table 2Patient characteristics in childhood ALL

Receptorlevel(sites/cell)

Glucocorticoid Receptors and Leukemia

these curves is significantly different from the other, andthe difference in the 3 groups compared together is highlysignificant (p < 0.001) with the use of the x2 test for trend.

High WBC at the time of initial presentation is wellcorrelated with a poor prognosis in ALL. For this reason weexamined remission duration and survival for the 21 patients with initial WBC in excess of 20.000. Effects ofreceptor level on remission duration for this subset ofpatients are shown in Chart 4. For either T- or null-cellpatients with blasts with less than 6.000 sites/cell, themedian remission duration was 8 months, and all patientshave relapsed. For the 7 patients with blasts with greaterthan 6,000 sites/cell (all null), the median remission duration is 25 months, and 3 of 7 remain in remission at 31, 66.and 74 months, respectively. Interestingly, the 2 null-cellgroups are equivalent with respect to age and WBC. Thus,the only identified difference between them is their receptorlevel.

Survival for this subset of patients with high initial WBCis shown in Table 3. As shown, T- and null-cell patients with

Table 3Survival and glucocorticoid receptors in "poor prognosis" ALL

(WBC > 20,000)

BfVSS'ON DURATION

Chart 3. Complete remission duration in patients with ALL as a functionof glucocorticoid receptor levels. A. patients with receptor levels 6000 glucocorticoid sites/cell; x. patients still in remission.

Receptor levels(sites/cell)31.0Proportion

alive1/91/5

4/7

Chart 4. Complete remission duration in patients with ALL and initialWBC above 20.000 cells/Cu mm. •¿�,T-cell patients with

M. E. Lippman et al.

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1978;38:4251-4256. Cancer Res Marc E. Lippman, Geraldine Konior Yarbro and Brigid G. Leventhal LeukemiaClinical Implications of Glucocorticoid Receptors in Human

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