[CANCER RESEARCH 32, 2524-2529, November 1972]

Lymphocyte Blastogenesis to Human Leukemia Cells and TheirRelationship to Serum Factors, Immunocompetence, andPrognosis1

Jordan U. Gutterman, Evan M. Hersh, Kenneth B. McCredie, Gerald P. Bodey, Sr., Victorio Rodriguez, andEmil J Freireich

Department of Developmental Therapeutics, The Universitv of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas77025

SUMMARY

Lymphocyte blastogenic studies to autologous leukemiacells were carried out in 35 patients with adult acute leukemia.The effects of serum on this response and correlation withstudies of general immunocompetence were studied.Twenty-five of 35 patients had positive blastogenesis. Ninepatients with acute myelogenous leukemia had complete orpartial abrogation of this response in autologous serum. Fivepatients had facilitation of the blastogenic response inautologous serum compared to the response in allogeneicserum. Patients with acute myelogenous leukemia had a morevigorous response to their leukemia cells than patients withacute lymphoblastic leukemia. Although studies of generalimmunocompetence tended to correlate with the in vitroresponse to leukemia cells, only one patient who wasunresponsive to autologous leukemia cells was immunoin-

competent. A good prognosis was correlated with a vigorousresponse to leukemia cells, inhibition or facilitation of thatresponse by autologous serum, and a persistently positiveblastogenic response in remission.

INTRODUCTION

Strong evidence exists that tumor-associated antigens arepresent in many, if not all, human cancers (15). Cell-mediatedimmune responses to membrane extracts of leukemia cellshave been reported by Oren and Herberman (18). A number ofworkers have also demonstrated that patients with acuteleukemia show an in vitro lymphocyte blastogenic response totheir own leukemia cells (8, 17, 19, 22). These studies havegenerally not evaluated the role of serum factors or correlatedthe results with the prognosis of the patient.

The current study was designed to evaluate critically therelationship between the presence of cell-mediated tumorimmunity and prognosis with particular regard to the role ofserum factors. In addition, the interrelationship betweengeneral immunocompetence and specific tumor responsivenesswas studied. The presence of a vigorous lymphocyteblastogenic response to leukemia cells, its inhibition or its

facilitation by autologous serum, and the persistence ofpositive in vitro lymphocyte responsiveness to leukemia cellswith time were found to correlate with a good prognosis.

MATERIALS AND METHODS

Thirty-five adult acute leukemia patients had studies ofblastogenic response to autologous leukemia cells in bothautologous and allogeneic serum.

Leukemic blast cells were collected from the peripheralblood of untreated patients on admission to the hospital. Thecells were collected with the IBM or the Aminco blood cellseparators as previously used to obtain lymphocytes (7). Thered blood cells were removed by exposure of the collectedcells (in their original acid-citrate-dextrose suspendingmedium) to 5 volumes of Tris-buffered ammonium chloride(2). After centrifugation and washing, the leukemia cells wereresuspended in Roswell Park Memorial Institute 1640 mediumwith 20% fetal calf serum. This suspension was mixed with a10% volume of dimethyl sulfoxide and frozen at l°/min to-120° in sterile glass ampuls at a leukocyte concentration of5 X 107/ml and in a volume of 1 ml. The ampuls were storedat —¿�180°in liquid nitrogen. On the day of study the ampulswere thawed rapidly at 37°, washed 3 times inSpinner-modified MEM2 (Hyland Laboratories, Los Angeles,

Calif.), and resuspended in MEM at room temperature. Thecells were counted in a hemocytometer chamber and viabilitywas determined by trypan blue dye exclusion (6).

For the lymphocyte culture and other lymphocyte studies,peripheral blood lymphocytes were collected, washed, andcultured as previously described (11). Cultures contained1 X IO6 washed lymphocytes, 1 ml of serum (either

autologous or allogeneic), 2 ml of MEM, and variousstimulants including leukemia cells, PHA (Difco Laboratories,Inc., Detroit, Mich.), SLO (Difco Laboratories, Inc.), andallogeneic normal leukocytes. The stimulator leukemia cellswere added at a dose range, in half-log intervals, varying fromIO4 to IO6; both unirradiated and irradiated (with 4,000 or

12,000 R) cells were used. Appropriate controls, consisting ofunstimulated lymphocytes, unirradiated leukemia cellscultured alone, irradiated leukemia cells cultured alone, and

1Supported by Grant ACS CI 22 from the American Cancer Societyand Grants CA 05831, FR 05511, and CA 11520 from NIH and theDepartment of Health, Education, and Welfare, USPHS, Bethesda, Md.

Received July 5, 1972; accepted August 10, 1972.

2The abbreviations used are: MEM, Eagle's minimal essential

medium; PHA, phytohemagglutinin; SLO, streptolysin O; SI,stimulation index; AML, acute myelogenous leukemia.

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Lymphocyte Blastogenesis to Human Leukemia Cells

irradiated leukemia cells cultured with irradiated lymphocytes,were set up. Cultures were incubated at 37°in an atmosphere

of 5% CO2 in air for 3 to 5 days (PHA, SLO) or for 7 days(mixed lymphocyte cultures and tumor cells). Harvesting wasaccomplished as previously described (11), by adding 2 /jCi ofthymidine-3H (Schwarz/Mann, Orangeburg, N. Y.; specificactivity, 1.9 Ci/mmole) for 3 hr and measuring the acid-insoluble radioactivity by liquid scintillation counting.

Lymphocyte blastogenesis was measured as the net cpm ofthymidine-3H incorporation controls subtracted from that of

the stimulated cultures. A 100% increase or 50% decrease inthymidine incorporation compared to the appropriate controlwas significant stimulation or inhibition at the 95% level ofconfidence (12) and a 300% increase or 75% decrease wassignificant at the 99% level of confidence. SI was defined asthe cpm of a stimulated culture divided by the cpm of theappropriate unstimulated culture.

Of the 35 adult patients, 24 had AML and 11 had acutelymphoblastic leukemia. Their median age was 28 (range 15 to71). There were 21 males and 14 females. Allchemotherapeutic regimens consisted of 5 days ofchemotherapy with intervals off therapy of at least 9 days.Regimens used were similar to those previously reported (14).The patients were studied as soon as their peripheral blood wasfree of leukemic cells, just prior to a course of chemotherapy,and at least 5 days after the completion of the previous course.The patients were studied prior to the 2nd or 3rd course ofremission induction therapy and again during the remission

maintenance therapy. Twenty-seven of the 35 patients had notbeen treated with chemotherapy previously, while 8 hadrelapsed from previous chemotherapeutic regimens. Thecriteria for remission have been described previously (16).

Statistical Analysis. Statistical analysis was performed bythe x2 or the Wilcoxon test.

RESULTS

Nineteen of 24 patients with AML and 6 of 11 with acutelymphoblastic leukemia had a positive blastogenic response(SI > 2) to autologous leukemia cells in autologous and/orallogeneic serum. Correlation between the degree ofblastogenic response to leukemia cells and the clinical responseis shown in Table 1. Seventeen of 20 (85%) patients withSI > 3 achieved remission. In contrast, only 60% of patientswith SI < 3 achieved a clinical remission. All 9 patients with aSI greater than 10 had a clinical remission. The vast majorityof patients with high Si's had AML. The remission duration

also correlated with the relapse rate. Thus, 78% of patientswith a SI less than 3 who achieved remission relapsedsubsequently. Their median duration of remission was only 19weeks. In contrast, only 24% of patients with a SI > 3 haverelapsed and none of the patients with a SI > 10 have relapsed.Their median duration of remission is greater than 21 weeks.Six of the 8 acute lymphoblastic leukemia patients achievingremission have relapsed in contrast to only 5 of the 18 withAML.

Table 1Correlation between degree of blastogenic response to leukemia cells and clinical response

No. in remission/no.testedSI<Si:

si;Si:C

3">3>

5> 10AML5/8

13/1612/138/8Acute

lymphoblasticleukemia4/7

4/43/31/1Total9/15

(60%)17/20(85%)15/16(94%)9/9 (100%)AML3/5

2/132/120/8No.

relapsed/no. inremissionAcute

lymphoblasticleukemia4/42/4

1/30/1Total7/9

(78%)4/17(24%)3/15 (20%)0/9 (0%)Median

durationof remission

(wk)a1920+

20+21+

a Difference between SI< 3, SI > 3, p = 0.02 (Wilcoxon); difference between SI < 3, SI > 10,p = 0.002 (Wilcoxon).

b Maximum SI in either autologous or allogeneic serum.

Table 2Correlation between hlastogenic response to leukemia cells, PHA, and SLO

Blastogenic response to

PHA SLO

SI Net cpm X IO3 SI Net cpm X IO3 SI

<3>3

>562(11-114)"84(13-148)69(13-145)55(13-145)139

(4.7-500)0322(13-1116)250(13-1116)464(13-1116)2.3

(0-46)c13.2(0-98)13.5 (0-98)17.7 (0-98)3.6(1.0-162)c

17.5 (1.5-843)14.7 (1.5-843)16.0(1.5-843)

a All values are medians with ranges in parentheses. Values represent maximum response in

autologous or allogeneic serum.b Difference in PHA response between SI< 3 and SI > 3 groups p = 0.05.c Difference in SLO response between SI< 3 and SI > 3 groups p = 0.013.

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Gutterman et al.

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The correlation between general immunocompetence asmeasured by the response to PHA and SLO and lymphocyteresponse to autologous leukemia cells is shown in Table 2. Theoverall median response to PHA in cpm was somewhat lowerin the group with SI< 3 compared to the group with SI > 3.The SI to PHA of the former group was lower than the latter.Of greater interest was the response to SLO in these groups ofpatients. Those with a SI to autologous leukemia cells above 3had a strikingly superior response to SLO (cpm) than thosewith a response less than 3. A similar pattern was noted for theSI to SLO. Despite this difference, only 1 of 15 patients with aSI to autologous leukemia cells less than 3 had a subnormalresponse to PHA (cpm, 20,000) (14). However, 7 of 15patients with SI < 3 had a subnormal response to SLO (cpm,< 2,000) (14) compared to 5 of 20 with a SI greater than 3.Thus, the proportion of patients considered immunoincompe-tent was only slightly greater in the group with a poorresponse to autologous leukemia cells. The overall response byAML and acute lymphoblastic leukemia patients to PHA wascomparable (median net cpm, 60 X IO3 and 67 X IO3,

respectively). Patients with AML had a somewhat morevigorous response to SLO than those with acute lymphoblasticleukemia (8.5 X IO3 compared to 3.7 X IO3 cpm,

respectively).The sera of 8 of the 25 patients with a positive blastogenic

response to autologous leukemia cells demonstrated aninhibitory effect on the blastogenic response compared to thatin allogeneic serum (Table 3). The blastogenic response wascompletely inhibited in 6 and was inhibited over 80% in theother 2. Seven of these 8 had AML, and 6 achieved aremission. Four of these were studied more than once and theinhibitory effect was constant in 3. The responses to PHA inautologous and allogeneic serum are noted in Table 3. Therewas no significant inhibition in autologous serum in terms ofcpm. However, there was inhibition in terms of the SI in 3 ofthe 8 patients, but in 2 of these it was less than 70%. Theresponse to SLO was not inhibited at all. Therefore, the seruminhibition was tumor specific in 7 of 8 patients.

The sera of 5 of the 25 patients with positive blastogenicresponses showed a facilitory effect on the blastogenicresponse to autologous leukemia cells (Table 4). There was asignificant increase in the tumor response of the lymphocytesof the patients when the lymphocytes were cultured inautologous serum rather than allogeneic serum. Four of the 5patients had AML and all achieved clinical remission. Threealso had significant facilitation of the response to PHA inautologous serum. Two of these and an additional patient alsohad an increased response to SLO in autologous serum. Thus,4 of 5 had an increased response to either PHA and/or SLO inautologous serum, and, therefore, the facüitory effect wasnonspecific.

Nineteen patients achieving a clinical remission had repeatstudies during the remission maintenance phase of theirtreatment. These results are shown in Table 5. Ten of 11(91%) of the patients who had a positive blastogenic responsein remission have continued in remission. In contrast, 7 of the8 patients who had a negative response to leukemia cellsduring remission have relapsed. Two of these patients werepositive initially (SI > 2). Five of 6 patients with persistentnegative blastogenic responses in remission have relapsed.

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Lymphocyte Blastogenesis to Human Leukemia Cells

Table 4Blastogenic response to autologous leukemia cells: serum facilitory effect

cpm XIO3Patient12345MedianDiagnosisAMLAM

LAMLAMLAcute

lymphoblasticleukemiaAutologous3.69.54.51.83.33.6Allogeneic1.080.09000.080.08SIAutologous5.759.780.912.910.012.9Allogeneic1.91.00.91.01.41.0ClinicalresponseR°RRRR0

R, remission.

Table 5Serial blastogenic response to leukemia cells

in patients achieving remission

Change in blastogenicresponseInitial

study Follow-upstudyPositive"

-> PositiveNegative -»PositivePositive

-»NegativeNegative -> NegativeChange

in clinicalstatusRemission-»

remission9

10

1Remission^

relapse1025

" Positive, SI > 2; negative, SI < 2.

The overall blastogenic responses to leukemia cells bylymphocytes of patients and lymphocytes of allogeneic normalsubjects cultured concurrently are shown in Table 6. Themedian response of AML patients to their own leukemia cellswas considerably greater than the response of acutelymphoblastic leukemia patients to their own cells(p< 0.005). The lymphocytes of normal subjects respondedto a greater degree to AML cells than to acute lymphoblasticleukemia cells, but this was not statistically significant.

DISCUSSION

Previous work from this laboratory has demonstrated thatimmunocompetence is associated with a favorable prognosis inpatients with acute leukemia (14). The present study hasidentified several new features which may be of relevance tothe prognosis of these patients. We have identified a strongcorrelation between the intensity of lymphocyteresponsiveness to autologous leukemia cells early in therapyand the likelihood of achieving a subsequent remission. Evenmore significant, the duration of remission is superior inpatients with vigorous responses to their leukemia cells. Someprevious studies have failed to identify this correlation.However, Oren and Herberman (18) found a correlationbetween a positive response to skin tests with autologousleukemia membranes and clinical remissions in acutelymphoblastic leukemia. It is interesting to speculate that thestrong immune response to leukemia cells may augment thechemotherapeutic effect and play a role in the remission

induction process and the overall prognosis of the patients.Similar speculations have been offered as an explanation forthe dramatic responses seen with chemotherapy in patientswith Burkitt's lymphoma (4) and choriocarcinoma (5).

This study has also shown the importance of serial studies oftumor immunity in patients with leukemia. The persistence ofa positive blastogenic response in remission or a change fromnegative to positive was associated with a good prognosis. Incontrast, patients who repeatedly had negative blastogenicresponse to their leukemia cells during remission or who havegone from positive to negative relapsed early. Therefore, thesetechniques should be used to evaluate patients on long-termchemotherapy and be used to guide future immunotherapytrials in acute leukemia.

Only 1 patient who failed to have a significant response toautologous tumor cells could be consideredimmunoincompetent by the response to PHA. In contrast,several patients unresponsive to leukemia cells had poorresponsiveness to the antigen SLO. Since macrophages arerequired for the in vitro lymphocyte responsiveness to antigens(13), one could speculate that perhaps impaired processing ofantigen by macrophages may be one of the reasons for lack oftumor cell responsiveness in individual patients. One pointagainst this view is the fact that several patients with vigorousresponse to tumor cells also had poor response to SLO in vitro.Hence, the evaluation of general immunocompetence does notinvariably parallel the in vitro response to tumor cells.

In addition to the general immunocompetence of theleukemia patient, another factor which may be of equalimportance for in vitro tumor responsiveness is theantigenicity of leukemia cells. Our data strongly suggest thatpatients with AML have a more intense immune response totheir own tumor cells compared to patients with acutelymphoblastic leukemia. As a group, there was no differencebetween AML patients and ALL patients in their response toPHA and only a slight difference in response to SLO. Theevidence that AML may be associated with more intenseimmune responses is further substantiated by the superiorresponse of allogeneic normal lymphocytes to AML cellscompared to acute lymphoblastic leukemia cells (although notsignificant statistically). The possibility that the AML cellsmay be more antigenic than acute lymphoblastic leukemiacells was also suggested by our earlier report that only AMLcells but not acute lymphoblastic leukemia cells have boundsurface immunoglobin which is presumed to be antibody (9).

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Gutterman et al.

Table 6Blastogenic response to leukemia cells

Net cpm X IO3 SI

PatientLymphocytesNormal

LymphocytesAM

L4.6°

(0-120.0)34.9

(0.937-104.0)Acute

lymphoblasticleukemia0.449

(0-3.35)11.1

(2.6-76.2)AML5.4

(0.9-1250)35.9

(1.6-579.0)Acute

lymphoblasticleukemia2.6

(0.7-12.1)17.7

(2.9-113)P0.060.35

" All values are medians with ranges in parentheses.

This suggests that there may be antigen deletion on some acutelymphoblastic leukemia cells. This has been suspected in apatient with malignant lymphoma (20) and in some animalleukemias (3). Alternatively, there may be antigen masking bysialic acid residues (23).

It has been previously recognized that patients with cancerhave factors in their serum which may inhibit cell-mediatedresponses to mitogens, antigens, and tumor cells. TheHellströms and coworkers have suggested that thetumor-specific blocking activity in patients with solid tumorsis due to either IgG antibody (10) or antigen-antibodycomplexes (21). In the present study, the inhibition of theresponsiveness of tumor cells in autologous serum was specificexcept in 1 instance. We have previously demonstrated that 7of 8 patients with AML who have inhibition of the blastogenicresponse by autologous serum have bound IgGimmunoglobulin on their cells (J. Gutterman, R. D. Rossen, W.T. Butler, K. B. McCredie, G. P. Bodey, E. J Freireich, and E.M. Hersh. Immunoglobulin on Tumor Cells and Tumor-induced Lymphocyte Blastogenesis in Human Acute Leukemia.New Engl. J. Med., in press). In contrast, only 1 of 16 patientswith a positive blastogenic response who did not demonstratean inhibitory effect had evidence for bound immunoglobulin.Although, a strong correlation between the presence ofantibody on leukemia cells and the blocking effect has beendemonstrated, our studies do not as yet clarify whether this isdue to antibody alone, to antigen-antibody complexes, or,perhaps, to other factors. In contrast to studies in solid tumorpatients where blocking of cell-associated responses isgenerally associated with progressive disease and a poorprognosis, our patients with this constellation of findingsappeared to have a relatively good prognosis as indicated bythe response to chemotherapy.

The demonstration of a serum facilitory effect in 5 patientswith acute leukemia demonstrates that the effects of serumfactors on in vitro cell-mediated immunity is complex andrequires careful investigation in individual patients with alltypes of cancers. The pathogenesis of this effect is unclear, butit appears to be associated with a good prognosis. None ofthese patients had demonstrable immunoglobulin to leukemiacells by direct immunofiuorescence. The possibility exists thatthere may be circulating antigen or antigen-antibodycomplexes in autologous serum which evokes an additiveblastogenic effect in vitro. We should also take into account

the previous report by Al Sarraf et al. (1) that lymphocyteresponse to PHA is reduced when incubated in allogeneicserum as compared to autologous serum. Therefore, ratherthan demonstrating autologous facilitation, we may be seeinginhibition by allogeneic serum. Additionally, the allogeneicserum in these cultures was pooled normal plasma, primarilyfrom females, many of whom had been pregnant. Therefore,we cannot rule out the possibility of nonspecific cytotoxicity.At any rate, the evaluation of serum factors in all studies ofcell-mediated immunity is of great importance in attemptingto understand the immune response to human tumors.

ACKNOWLEDGMENTS

The authors wish to acknowledge the excellent technical assistanceof Mrs. Carol Hunter and Mrs. Annette Matthews. We also wish tothank Mrs. Gloria Walker for preparation of the manuscript.

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1972;32:2524-2529. Cancer Res   Jordan U. Gutterman, Evan M. Hersh, Kenneth B. McCredie, et al.   PrognosisRelationship to Serum Factors, Immunocompetence, and Lymphocyte Blastogenesis to Human Leukemia Cells and Their

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