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T-cell markers in breast cancer patients at diagnosis
CLINICAL
IMMUNOLOGY
AND
T-Cell
12, 396403
IMMUNOPATHOLOGY
Markers
(1979)
in Breast Cancer Diagnosis’
Patients
at
JEAN M. GOUST, BETTY S. ROOF, H. HUGH FUDENBERG, PAUL H. O’BRIEN Department
of Surgery,
of Basic Medical
and University
Clinical
Immunology
of South
and
Carolina,
Microbiology. Charleston,
and South
AND Department
Carolina
29403
Received August 2. 1978 The number of circulating lymphocytes, the percentage and number of active (TEa) and total (TEt) rosette-forming cells, andthe percentageof rosettesformedbetween patients’ T cells and cells of the B-lymphoblastoid cell line PGLC-33H (PGLC-R) were determined 2-4 weeks after diagnosis in 68 patients with breast cancer. Significant lymphopenia wasfound in stagesIII and IV. The percentageof TEt was decreased in stage IV only. The percentage of TEa, in contrast, was at least 1 SD above the mean control value in 66% of stage I patients, and the percentages of patients with absolute numbers of TEa in the peripheral blood 1 SD or more below the normal range (688 ? 159/mm3) were 25% in stage I, 47% in stage II, 73% in stage III, and 84% in stage IV. These data show a suggestive parallel with previously reported rates of relapses for stages I-IV andindicatethat highlevelsof TEa may be associated with a better prognosis. PGLC-R were also decreased in stages I-IV compared with controls, with greater
decreases corresponding to moreadvancedstages of the disease. Incubationof patients’ serumwith normallymphocytesinduceda smalldecrease in TEt but did not affectTEa or PGLC-R.The prognosticsignificance of TEa levelsandthe valueof T-cell markers studies for the optimal use of immunotherapy
are discussed.
INTRODUCTION Decreased cell-mediated immune responses have been demonstrated in a variety of neoplastic diseases(l-5). In breast cancer patients, both a functional defect in lymphocyte response to mitogens and a decrease in the number of T lymphocytes reacting with sheep red blood cells (SRBC) have been described; however,
the latter may have been underestimated, since in most studies only percentages of T cells have been determined, without taking into account a possible coexisting lymphopenia (6,7). It is also not unlikely, as suggested by Wybran and Fudenberg (8) and by Whitehead et al. (5), that this deficiency could selectively affect some subpopulation of T lymphocytes. In the present investigation, various T-cell markers were studied in breast cancer patients shortly after diagnosis of various stages of the disease. The percentage and total number of total T cells forming rosettes with SRBC (TEt) were determined, as well as the subpopulation of active T cells (TEa), which is thought to play an important role in immunosurveillance (8, 9). Another T-cell marker which reacts with B-lymphoblastoid cell lines (LCL) producing IgM (IO) was also studied; we have shown previously that this marker is decreased in multiple ’ This is publication No. 238 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by USPHS Grant AI-13484 and by the South Carolina State Allocation for Biomedical Research (GR-87). 3% 0090-1229/79/0403%-08$01.00/O Copyright All rights
@I 1979 by Academic Press. Inc. of reproduction in any form resewed.
T-CELL
sclerosis in association complexes (11).
397
MARKERS IN BREAST CANCER
with increased
levels of circulating
antigen-antibody
PATIENTS AND METHODS
Patients. Sixty-eight patients were studied after surgical diagnosis, at least 2 weeks after surgery and prior to chemotherapy or radiotherapy. Staging was determined according to the TNM classification (International Union Against Cancer): stage I, Tl-2NOMO; stage II, Tl-2NlMO; stage III, Tl-4NO-2MO; stage IV, Tl-4NO-3Ml. The number of patients, mean age, and ethnic origin in each group are listed in Table 1. Until completion of the study, the personnel performing the assays had no knowledge of the clinical status of the patients, nor the clinicians of the laboratory results. Age- and sex-matched controls were studied simultaneously. For each series of tests, 20 ml of heparinized blood and 10 ml of blood without anticoagulant were drawn separately. Proper informed consent was obtained from each patient. Mononuclear cells (MNC). The heparinized blood was mixed with Hanks’ balanced salt solution (HBSS), pH 7.4, and centrifuged on Ficoll--1sopaque for 35 min at 400g. The MNC layer was washed three times with HBSS and resuspended at a final concentration of 2 x 10’ cells/ml. Rosetting with sheep red blood cells. SRBC, obtained weekly from a single, selected sheep maintained at the Medical University of South Carolina, were washed three times in Alsever’s solution and resuspended to final SRBC:MNC ratios of 30: 1 for total rosettes (TEt) and 8: 1 for active rosettes (TEa) (9). After addition of 0.1 ml of SRBC to 0.05 ml of MNC, the tubes were centrifuged for 5 min at l5Og . TEa were counted immediately; TEt were determined after overnight incubation at 4°C. For both tests, the percentage of MNC surrounded by three or more SRBC was determined by counting 200 MNC in a hemocytometer. The absolute numbers of TEa and TEt were determined by calculation of the corresponding total and differential leukocyte counts. For the 68 normal controls used in this study the values (mean + SD)-were 30.4 2 7.3% (688 + 159/mm3) for TEa and 70.1 t 7.8% (1468 k 427/mm3) for TEt; the number of total MNC was 2256 + 440/mm3. Rosette formation between MNC and the B-lymphoblastoid cell line PGLC-33H (PGLC-R). The technique of Jondal et al. (10) was used. The IgM-secreting cell line PGLC-33H, obtained through the courtesy of Dr. P. R. Glade (Department of Pediatrics, Miami School of Medicine), was maintained in stationary culture in minimal essential medium (MEM) supplemented with 20% heat-inactivated fetal TABLE BREAST
CANCER Age
Patients Stage Stage Stage Stage
I II III IV
No. 12 17 19 20
Mean 58 52 52 58
1
PATIENTS
STUDIED
Ethnic origin
(yews)
Range 37-74 37-73 30-74 29-76
Caucasian 7 13
11 11
Negro 5 4 8 9
398
GOUST
ET
AL.
calf serum (FCS). The concentration optimal for the assay was found to be from 7.5 x lo5 to 1.0 x 10” cells/ml. On the day of the tests, cells were harvested, washed three times with MEM then resuspended in MEM plus 20% FCS at a concentration of 4 x lO’/ml. Only cells whose viability was higher than 90% by trypan blue exclusion test were used. MNC were resuspended at 2 x 10Vml in MEM plus 20% FCS. Aliquots (0.050 ml) of the two cell suspensions were mixed, centrifuged at 4°C for 5 min at 15Og, then incubated for 1 hr in an ice-water bath. After gentle resuspension, 0.050 ml of 2.5% cold glutaraldehyde was added in order to stop the reaction if more than five tests had to be read. No differences in the final results were found whether or not glutaraldehyde was added at this stage. The percentage of PGLC-R (defined as a PGLC-33H cells surrounded by three or more MNC) was determined in duplicate in each instance. For the controls used in this study, the percentage was 29.6 + 8.6% (mean t SD), in agreement with the results of Jondal et al. (10) for IgM-producing human lymphoblastoid cell lines. Effect of breast cancer sera on rosette formation by normal lymphocytes. This procedure was essentially similar to the one described by Whitehead et al. (5). Sera from breast cancer patients after determination of TEa, TEt, and PGLC-R were first absorbed with packed RBC from the lymphocyte donor in 1: 1 (v:v) ratio for 2 hr at 4”C, with intermittent shaking. Serum (1.5 ml) was then incubated with 0.5 ml of lymphocytes (5 x lo6 cells: 0.075 ml of serum for lo5 lymphocytes) for 2 hr at 4°C in a 15-ml sterile plastic tissue culture tube (Falcon No. 2040) and then washed three times with HBSS. These normal lymphocytes were then tested for rosette formation by the procedures described above. RESULTS
Lymphopenia in Breast Cancer Patients As shown in Tables 2 and 3, there was a decrease in the number of circulating lymphocytes in breast cancer patients, the degree thereof depending upon the stage. Since the time between surgical diagnosis and test was from 2 weeks to 2 PERIPHERAL
BLOOD PATIENTS
TABLE 2 LYMPHOCYTES. TEa. TEt. AND PGLC-R IN BREAST 2-6 WEEKS AFTER DIAGNOSIS AND IN CONTROLS Breast
Normal controls Lymphocytes* TEa (%)” (Absolute)” TEt (%)” (Absolute)* PGLC-R’
Stage
038)”
Cells 2256 30.4 688 70.1 1468 29.6
f 2 t + t -c
640’ 7.3 159 7.8 527 8.6
I
(12) 2246 41.1 911 66.5 1437 20.5
+ zk + +-t f
1004 10.6 521 7.0 750 13
” Number of patients. b Total number per millimeter cubed of peripheral e Mean -t SD. rl Percentage of total mononuclear cells. p Percentage of PGLC cells binding three or more
cancer
Stage II (17) 1608 36.05 587 71.3 1149 22.4
c t2 +2 t
693 9.5 309 8.7 504 9.4
blood.
lymphocytes.
CANCER
patients Stage III (19) 1524 28.2 475 66.3 1102 13.6
i+ f ? ? +
713 9.8 304 10.0 548 11.0
Stage IV (20) 1404 30.4 414 63.9 869 14.5
t _f zk 2 t +
614 9.8 230 12.15 361 10.7
T-CELL
MARKERS
IN BREAST TABLE
BREAST
VARIATIONS IN LEVELS CANCER PATIENTS 2-6
OF PERIPHERAL WEEKS AVER
Lymphocytes’ Patients
399
CANCER
3 LYMPHOCYTES, TEa, AND TEt IN RELATIVE TO NORMAL CONTROL
BLOOD DIAGNOSIS
TEa (absolute)”
TEa (%)b
VALUES
TEt (o/o)*
TEt (absoluteY’
He
N
L
H
N
L
H
N
L
~H
81
668
253
668
81
253
587
425
-0
253
415
334
I 6
5 29
11 65
S 30
4 23
8 47
7 41
8 47
If
i
5;
7 41
2;
9 64
:
-0
326
68 13
:
224
73 14
153
10 54
3;
”
448
10 56
-0
14 78
2;
1 5
3 15
16 80
2 10
1 6
15 84
5 22
8 48
6 30
-
3 15
17 85
1 5
15 65
6 30
N
L
H
N
L
81
668
2:
Stage I ( 12)d %’ No.’ stage II (17) No. % Stage III (19) % NO. Stage IV (20) No. %
0
a Total number per millimeter cubed of peripheral blood. b Percentage of total mononuclear cells. r H, =I SD above mean control value: N. within 1 SD of normal conlrol value; L, 31 SD below normal control value. d Number of patients. e Number of patients with values in the range indicated. ’ Percentage of patients with values in the range indicated.
months, regardless of the stage of the disease, this lymphopenia cannot be explained by the surgical procedure alone. The decrease was progressive and almost linear (u = 0.89), the nadir being reached in stage IV breast cancer patients, who had 38% fewer lymphocytes than normal controls. Percentage Versus Absolute Numbers of TEt and TEa The percentages of both TEa and TEt did not differ from normal controls at any stage, except for an increase in TEa in stage I @ < 0.05); however, a small percentage of patients at each stage had lower percentages than normal controls (Table 3). On the other hand, the absolute numbers of both TEa and TEt differed significantly from the controls. The decrease in TEt was almost linear (I = 0.96) from stage I to IV, showing a trend almost identical to the decrease in MNC. This explains the fact that the percentage of TEt does not change significantly in breast cancer patients regardless of the stage of the disease. A similar but less striking decrease was seen for TEa in patients at stages II, III, and IV. In addition, 8 out of 12 patients in stage I had an absolute number of TEa at least 1 SD above control values. The overall results indicate that, in this group, TEa were 28% above the mean value for the control group. This was due to both a near normal number of circulating lymphocytes and an increased percentage of TEa. PGLC Rosettes The percentage of PGLC-R (Fig. 1) was significantly lower than control values at every stage of the disease: 8/12 (66%) in stage I, 7/13 (53%) in stage II, 13117 (76%) in stage III, and 12/18 (66%) in stage IV had PGLC-R 1 SD or more below control values. This decrease was not correlated with the decrease in the absolute number of lymphocytes or TEt or with the changes in TEa values.
400
GOUST
C
SI
ET
AL.
SII
Sill
SIV
FIG. 1. Percentages of PGLC-R in the control group (C), and in breast cancer patients tested 2-4 weeks after diagnosis in stages I (SI), II (SII), III (SIIK), and IV (SW). Bars indicate mean ? standard deviation.
Effect of Breast Cancer Sera on Rosette Formation by Normal Lymphocytes Sera from patients at various stages (whose TEt, TEa, and PGLC-R levels were determined on the day the serum was obtained) induced a small (15 2 4.8%) but significant (p < 0.05) decrease in TEt from normal donors, regardless of whether or not their own TEt were decreased (Table 4). TEa and PGLC-R remained unchanged. DISCUSSION
Previous studies of circulating T cells in breast cancer patients have shown decreases in the percentage of T cells (12, 13), associated with near normal or slightly increased percentages of B cells. However, as described in the present work lymphopenia is frequent in breast cancer and is more marked in patients at advanced stages of the disease. This is in agreement with the data of Glas et al. (6). The time between the beginning of the symptoms and surgery, limited to biopsy in stage IV, was frequently unknown; however, the patients in this study were first tested from 2 to 6 weeks after surgery, thus eliminating a possible cause of exogenous immunodepression. Hence, the lymphopenia at the time of testing could be considered as a first symptom of abnormal immune response. Therefore, it is necessary to take into account not only the percentages of various T-cell
T-CELL
MARKERS
IN
BREAST
TABLE CHANGES
IN
TEt, TEa,
AND WITH
NONSPECIFIC
SERA FROM
Value for
4 T
SUPPRESSION
11 BREAST
Normal control lymphocytes
70.5 k 3c
Lymphocytes from Patient 1 Normal lymphocytes incubated with serum from Patient 1
76.5 44
Lymphocytes from Patient 3 Normal lymphocytes incubated with serum from Patient 3
(6W 57 55 (78) 52 59 (83)
(PGLC-R)
AFTER
INCUBATION
PATIENTS
TEa (%)” 34.7 k 0.7 48.5 10.5 (30) 30 32 (94) 33 38 (111) 40 38.5
PGLC-R (%)” 30.0 * 0.5 33.5 (111) 24
630) 32.5 (32.5)
Lymphocytes from Patient 4 Normal lymphocytes incubated with serum from Patient 4
(t;)
(102)
(80)
Lymphocytes from Patient 5 Normal lymphocytes incubated with serum from Patient 5
77 56 (7%
45.5 35
30 24
(102)
(89
Lymphocytes from Patient 6 Normal lymphocytes incubated with serum from Patient 6
63 65 (92) 60 70.5 (109 47 62
49 34 (109 50 32.5 (104) 34 29
15 30 (109
(88)
(85)
Lymphocytes from Patient 7 Normal lymphocytes incubated with serum from Patient 7 Lymphocytes from Patient 8 Normal lymphocytes incubated with serum from Patient 8
74.5
CELLS
CANCER
TEt (%)”
Lymphocytes from Patient 2 Normal lymphocytes incubated with serum from Patient 2
401
CANCER
Lymphocytes from Patient 9 Normal lymphocytes incubated with serum from Patient 9
55 72 (102)
Lymphocytes from Patient 10 Normal lymphocytes incubated with serum from Patient 10
73 74 (104) 31.5 64
Lymphocytes from Patient 11 Normal lymphocytes incubated with serum from Patient 11
31 (lit) 40 32.5 (95) 25.5 35.5 (104)
19 31.5
23 39
(126) 10 29.5 (98.3) 10 39 (139 9 36.5
(121) -
n Percentage of total mononuclear cells. b Percentage of PGLC cells binding three or more lymphocytes. c Mean * SD. d Percentage of mean value for normal lymphocytes without incubation.
subsets but also millimeter cubid in this study the except stage IV,
their absolute numbers, expressed as the number of rosettes per of blood, as discussed previously by Kerman et al. (14). Indeed, percentage of TEt was near normal in almost all patient groups in contrast with the results of Keller et al. (12) and Whitehead et
402
GOUST
ET
AI..
al. (5); however, when the absolute number of TEt rosettes was calculated, a progressive decrease became obvious, which strictly paralleled the decrease in lymphocytes. One can thus consider that T cells, as a whole. remain in normal proportion within the circulation, although the more advanced the disease is at the time of diagnosis, the more their total numbers are decreased. In this study we found a decrease of TEt percentages in only a few patients, indicating that the receptors for sheep erythrocytes were not masked on the cells from most patients. This may be due to our extensive washing of the cells before rosette tests were performed, removing an inhibitor (5), which might be antigen-antibody complexes causing steric hindrance. Such an inhibitor, if present, apparently did not affect active rosette-forming cells (TEa) in this study nor the population of PGLC rosette-forming cells. The increased percentage of TEa in stage I and, to a lesser extent, in stage II breast cancer patients suggests a possible prognostic significance. These cells have been shown to proliferate actively in the peripheral blood (15) and be involved in T-cell-mediated cytotoxicity (16), and their role in immunosurveillance in cancer patients has been emphasized (8, 9). Their increase in stage I may indicate an ongoing immune response against the tumor. However, further observation is needed to determine whether patients with high TEa in stage I may have a relatively longer disease-free interval after surgery, whereas those with low TEa would require immunotherapy. On the other hand, it has been reported in one clinical study (17) that the percentages of relapse 5 years after diagnosis were respectively 23% for patients without lymph node involvement. 49% when one to three nodes were involved, and 81% when four or more nodes were involved. It is noteworthy that the percentage of patients in the present study with absolute numbers of TEa 1 SD or more below normal values was, at each stage, very close to the 5-year recurrence rate previously reported. Longitudinal studies have recently been initiated in this laboratory to ascertain whether stage I and II patients with high TEa levels have a normal life span whereas those with low TEa may develop metastases within a few years after surgical removal of the primary tumor. The binding of peripheral blood T cells to B-LCL occurs through receptors which have not been identified yet, but it apparently does not involve Fc or E-rosette receptors. Our studies of this phenomenon (J. M. Goust, manuscript in preparation) show that only two-thirds of normal peripheral blood T cells and thymocytes are able to bind to LCL. It is, therefore, possible that these T cells may belong to a specific subset, possibly involved in T-B cell interactions, as suggested by Jondal er al. (10). It might have been more logical in this study to express the data as percentage of MNC bound to LCL. However, in patients with a low percentage of PGLC-R, the percentage of MNC bound to B-LCL was consistently lower than the value observed in normal controls, and it is technically easier to count the percentage of rosetted B-LCL than to determine the percentage of free and bound MNC. The reduction in the subset of T cells binding to this cell line in advanced disease may reflect either the selective masking of a T-cell receptor or the disappearance of a specific subset bearing the receptor, since it is not correlated with decreases in other subsets of T cells. In a previous study (11). we found that 70% of multiple sclerosis patients had both decreased PGLC-R and
T-CELL
MARKERS
IN
BREAST
CANCER
403
increased immune complexes, assessed by the Clq-binding technique. A similar inverse correlation could exist in breast cancer. Indeed, Rossen et al. (18) have reported increased Clq binding in 74% of breast cancer patients, with the highest values obtained in patients who had active disease. It is possible, therefore, that the disappearance of PGLC rosette-forming T cells may allow the production of antibodies to an etiologic agent(s), followed by immune complex formation, which in turn would lead to further impairment of cell-mediated immune responses. ACKNOWLEDGMENTS The help of M. Keels, R. N., in collecting blood samples and clinical information is gratefully acknowledged. The reliable and skillful technical assistance of Gena Ciabattari has been invaluable for this work. We thank Charles L. Smith for excellent editorial assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Catalona, W. J., Sample, W. F., and Chretien, B., Cancer 31, 65-71 (1973). Dellon, A. L. Potvin, C., and Chretien, P. B. Cancer 35, 687-694 (1975). Stefani S. S., Kerman, R., and Abbate, J., Amer. J. Roentgenol. 126, 880-886 (1976). Stefani, S. S., Kerman, R., and Abbate, J., Cancer 37, 2792-2796 (1976). Whitehead, R. H., Roberts, G. P., Thatcher, J., Teasdale, C., and Hughes, L. E., J. Nut. Cancer Inst. 58, 1573-1576 (1977). Glas, U., Wasserman, J., Blomgren, H., and DeSchryver, A.,lnr. J. Radiat. Oncol. Biol. Phys. 1, 189- 195 (1976). Stjernsward, J., Jondal, M., Vanky, F.. Wigzell, H., and Sealy, R., Lancet 1, 1352-1365 (1972). Wybran, J., and Fudenberg, H. H., In “Clinical Tumor Immunology” (J. Wybran and M. Staquet, Eds.), pp. 31-40, Pergamon, London (1976). Wybran, J., and Fudenberg, H. H., J. Clin. Invest. 52, 1026-1031 (1973). Jondal, M., Klein, M., and Yeyenoff, E., Stand. J. Immunol. 4, 259-266 (1975). Goust, J. M., Carnes, J. E., Chenais, F., Hames, C. G., Fudenberg, H. H., and Hogan, E. L., Neurology 28, 421-425 (1978). Keller, S. E., Ioachim, H. L., Pearse, T., and Siletti, D. M., Amer. J. C/in. Pathol. 65,445-449 (1976). Nemoto, T., Han, T., and Minowada, J., J. Nut. Cancer Inst. 53, 64-645 (1974). Kerman, R. H., Smith, R., Stefani, S. S., and Ezdinli, Z., Cancer Res. 36, 3274-3278 (19761. DeBruyere, M., Lachapelle, J. M., and Anguita, T., Int. Arch. Allergy Appl. Immunol. 54,50-57 (1977). Wybran, J., Hellstrom, I., Hellstrom, K. E., and Fudenberg, H. H., Int. J. Cancer 13, 515-521 (1974). Fisher, B., Black. N. H., and Brose, I. D. J., Cancer 2, 1071-1080 (1969). Rossen, R. D.. Reisberg, M. A., Hersh, E. M., and Gutterman, J. V., J. Nat. Cancer Inst. 58, 1205-1215 (1977).
IMMUNOLOGY
AND
T-Cell
12, 396403
IMMUNOPATHOLOGY
Markers
(1979)
in Breast Cancer Diagnosis’
Patients
at
JEAN M. GOUST, BETTY S. ROOF, H. HUGH FUDENBERG, PAUL H. O’BRIEN Department
of Surgery,
of Basic Medical
and University
Clinical
Immunology
of South
and
Carolina,
Microbiology. Charleston,
and South
AND Department
Carolina
29403
Received August 2. 1978 The number of circulating lymphocytes, the percentage and number of active (TEa) and total (TEt) rosette-forming cells, andthe percentageof rosettesformedbetween patients’ T cells and cells of the B-lymphoblastoid cell line PGLC-33H (PGLC-R) were determined 2-4 weeks after diagnosis in 68 patients with breast cancer. Significant lymphopenia wasfound in stagesIII and IV. The percentageof TEt was decreased in stage IV only. The percentage of TEa, in contrast, was at least 1 SD above the mean control value in 66% of stage I patients, and the percentages of patients with absolute numbers of TEa in the peripheral blood 1 SD or more below the normal range (688 ? 159/mm3) were 25% in stage I, 47% in stage II, 73% in stage III, and 84% in stage IV. These data show a suggestive parallel with previously reported rates of relapses for stages I-IV andindicatethat highlevelsof TEa may be associated with a better prognosis. PGLC-R were also decreased in stages I-IV compared with controls, with greater
decreases corresponding to moreadvancedstages of the disease. Incubationof patients’ serumwith normallymphocytesinduceda smalldecrease in TEt but did not affectTEa or PGLC-R.The prognosticsignificance of TEa levelsandthe valueof T-cell markers studies for the optimal use of immunotherapy
are discussed.
INTRODUCTION Decreased cell-mediated immune responses have been demonstrated in a variety of neoplastic diseases(l-5). In breast cancer patients, both a functional defect in lymphocyte response to mitogens and a decrease in the number of T lymphocytes reacting with sheep red blood cells (SRBC) have been described; however,
the latter may have been underestimated, since in most studies only percentages of T cells have been determined, without taking into account a possible coexisting lymphopenia (6,7). It is also not unlikely, as suggested by Wybran and Fudenberg (8) and by Whitehead et al. (5), that this deficiency could selectively affect some subpopulation of T lymphocytes. In the present investigation, various T-cell markers were studied in breast cancer patients shortly after diagnosis of various stages of the disease. The percentage and total number of total T cells forming rosettes with SRBC (TEt) were determined, as well as the subpopulation of active T cells (TEa), which is thought to play an important role in immunosurveillance (8, 9). Another T-cell marker which reacts with B-lymphoblastoid cell lines (LCL) producing IgM (IO) was also studied; we have shown previously that this marker is decreased in multiple ’ This is publication No. 238 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by USPHS Grant AI-13484 and by the South Carolina State Allocation for Biomedical Research (GR-87). 3% 0090-1229/79/0403%-08$01.00/O Copyright All rights
@I 1979 by Academic Press. Inc. of reproduction in any form resewed.
T-CELL
sclerosis in association complexes (11).
397
MARKERS IN BREAST CANCER
with increased
levels of circulating
antigen-antibody
PATIENTS AND METHODS
Patients. Sixty-eight patients were studied after surgical diagnosis, at least 2 weeks after surgery and prior to chemotherapy or radiotherapy. Staging was determined according to the TNM classification (International Union Against Cancer): stage I, Tl-2NOMO; stage II, Tl-2NlMO; stage III, Tl-4NO-2MO; stage IV, Tl-4NO-3Ml. The number of patients, mean age, and ethnic origin in each group are listed in Table 1. Until completion of the study, the personnel performing the assays had no knowledge of the clinical status of the patients, nor the clinicians of the laboratory results. Age- and sex-matched controls were studied simultaneously. For each series of tests, 20 ml of heparinized blood and 10 ml of blood without anticoagulant were drawn separately. Proper informed consent was obtained from each patient. Mononuclear cells (MNC). The heparinized blood was mixed with Hanks’ balanced salt solution (HBSS), pH 7.4, and centrifuged on Ficoll--1sopaque for 35 min at 400g. The MNC layer was washed three times with HBSS and resuspended at a final concentration of 2 x 10’ cells/ml. Rosetting with sheep red blood cells. SRBC, obtained weekly from a single, selected sheep maintained at the Medical University of South Carolina, were washed three times in Alsever’s solution and resuspended to final SRBC:MNC ratios of 30: 1 for total rosettes (TEt) and 8: 1 for active rosettes (TEa) (9). After addition of 0.1 ml of SRBC to 0.05 ml of MNC, the tubes were centrifuged for 5 min at l5Og . TEa were counted immediately; TEt were determined after overnight incubation at 4°C. For both tests, the percentage of MNC surrounded by three or more SRBC was determined by counting 200 MNC in a hemocytometer. The absolute numbers of TEa and TEt were determined by calculation of the corresponding total and differential leukocyte counts. For the 68 normal controls used in this study the values (mean + SD)-were 30.4 2 7.3% (688 + 159/mm3) for TEa and 70.1 t 7.8% (1468 k 427/mm3) for TEt; the number of total MNC was 2256 + 440/mm3. Rosette formation between MNC and the B-lymphoblastoid cell line PGLC-33H (PGLC-R). The technique of Jondal et al. (10) was used. The IgM-secreting cell line PGLC-33H, obtained through the courtesy of Dr. P. R. Glade (Department of Pediatrics, Miami School of Medicine), was maintained in stationary culture in minimal essential medium (MEM) supplemented with 20% heat-inactivated fetal TABLE BREAST
CANCER Age
Patients Stage Stage Stage Stage
I II III IV
No. 12 17 19 20
Mean 58 52 52 58
1
PATIENTS
STUDIED
Ethnic origin
(yews)
Range 37-74 37-73 30-74 29-76
Caucasian 7 13
11 11
Negro 5 4 8 9
398
GOUST
ET
AL.
calf serum (FCS). The concentration optimal for the assay was found to be from 7.5 x lo5 to 1.0 x 10” cells/ml. On the day of the tests, cells were harvested, washed three times with MEM then resuspended in MEM plus 20% FCS at a concentration of 4 x lO’/ml. Only cells whose viability was higher than 90% by trypan blue exclusion test were used. MNC were resuspended at 2 x 10Vml in MEM plus 20% FCS. Aliquots (0.050 ml) of the two cell suspensions were mixed, centrifuged at 4°C for 5 min at 15Og, then incubated for 1 hr in an ice-water bath. After gentle resuspension, 0.050 ml of 2.5% cold glutaraldehyde was added in order to stop the reaction if more than five tests had to be read. No differences in the final results were found whether or not glutaraldehyde was added at this stage. The percentage of PGLC-R (defined as a PGLC-33H cells surrounded by three or more MNC) was determined in duplicate in each instance. For the controls used in this study, the percentage was 29.6 + 8.6% (mean t SD), in agreement with the results of Jondal et al. (10) for IgM-producing human lymphoblastoid cell lines. Effect of breast cancer sera on rosette formation by normal lymphocytes. This procedure was essentially similar to the one described by Whitehead et al. (5). Sera from breast cancer patients after determination of TEa, TEt, and PGLC-R were first absorbed with packed RBC from the lymphocyte donor in 1: 1 (v:v) ratio for 2 hr at 4”C, with intermittent shaking. Serum (1.5 ml) was then incubated with 0.5 ml of lymphocytes (5 x lo6 cells: 0.075 ml of serum for lo5 lymphocytes) for 2 hr at 4°C in a 15-ml sterile plastic tissue culture tube (Falcon No. 2040) and then washed three times with HBSS. These normal lymphocytes were then tested for rosette formation by the procedures described above. RESULTS
Lymphopenia in Breast Cancer Patients As shown in Tables 2 and 3, there was a decrease in the number of circulating lymphocytes in breast cancer patients, the degree thereof depending upon the stage. Since the time between surgical diagnosis and test was from 2 weeks to 2 PERIPHERAL
BLOOD PATIENTS
TABLE 2 LYMPHOCYTES. TEa. TEt. AND PGLC-R IN BREAST 2-6 WEEKS AFTER DIAGNOSIS AND IN CONTROLS Breast
Normal controls Lymphocytes* TEa (%)” (Absolute)” TEt (%)” (Absolute)* PGLC-R’
Stage
038)”
Cells 2256 30.4 688 70.1 1468 29.6
f 2 t + t -c
640’ 7.3 159 7.8 527 8.6
I
(12) 2246 41.1 911 66.5 1437 20.5
+ zk + +-t f
1004 10.6 521 7.0 750 13
” Number of patients. b Total number per millimeter cubed of peripheral e Mean -t SD. rl Percentage of total mononuclear cells. p Percentage of PGLC cells binding three or more
cancer
Stage II (17) 1608 36.05 587 71.3 1149 22.4
c t2 +2 t
693 9.5 309 8.7 504 9.4
blood.
lymphocytes.
CANCER
patients Stage III (19) 1524 28.2 475 66.3 1102 13.6
i+ f ? ? +
713 9.8 304 10.0 548 11.0
Stage IV (20) 1404 30.4 414 63.9 869 14.5
t _f zk 2 t +
614 9.8 230 12.15 361 10.7
T-CELL
MARKERS
IN BREAST TABLE
BREAST
VARIATIONS IN LEVELS CANCER PATIENTS 2-6
OF PERIPHERAL WEEKS AVER
Lymphocytes’ Patients
399
CANCER
3 LYMPHOCYTES, TEa, AND TEt IN RELATIVE TO NORMAL CONTROL
BLOOD DIAGNOSIS
TEa (absolute)”
TEa (%)b
VALUES
TEt (o/o)*
TEt (absoluteY’
He
N
L
H
N
L
H
N
L
~H
81
668
253
668
81
253
587
425
-0
253
415
334
I 6
5 29
11 65
S 30
4 23
8 47
7 41
8 47
If
i
5;
7 41
2;
9 64
:
-0
326
68 13
:
224
73 14
153
10 54
3;
”
448
10 56
-0
14 78
2;
1 5
3 15
16 80
2 10
1 6
15 84
5 22
8 48
6 30
-
3 15
17 85
1 5
15 65
6 30
N
L
H
N
L
81
668
2:
Stage I ( 12)d %’ No.’ stage II (17) No. % Stage III (19) % NO. Stage IV (20) No. %
0
a Total number per millimeter cubed of peripheral blood. b Percentage of total mononuclear cells. r H, =I SD above mean control value: N. within 1 SD of normal conlrol value; L, 31 SD below normal control value. d Number of patients. e Number of patients with values in the range indicated. ’ Percentage of patients with values in the range indicated.
months, regardless of the stage of the disease, this lymphopenia cannot be explained by the surgical procedure alone. The decrease was progressive and almost linear (u = 0.89), the nadir being reached in stage IV breast cancer patients, who had 38% fewer lymphocytes than normal controls. Percentage Versus Absolute Numbers of TEt and TEa The percentages of both TEa and TEt did not differ from normal controls at any stage, except for an increase in TEa in stage I @ < 0.05); however, a small percentage of patients at each stage had lower percentages than normal controls (Table 3). On the other hand, the absolute numbers of both TEa and TEt differed significantly from the controls. The decrease in TEt was almost linear (I = 0.96) from stage I to IV, showing a trend almost identical to the decrease in MNC. This explains the fact that the percentage of TEt does not change significantly in breast cancer patients regardless of the stage of the disease. A similar but less striking decrease was seen for TEa in patients at stages II, III, and IV. In addition, 8 out of 12 patients in stage I had an absolute number of TEa at least 1 SD above control values. The overall results indicate that, in this group, TEa were 28% above the mean value for the control group. This was due to both a near normal number of circulating lymphocytes and an increased percentage of TEa. PGLC Rosettes The percentage of PGLC-R (Fig. 1) was significantly lower than control values at every stage of the disease: 8/12 (66%) in stage I, 7/13 (53%) in stage II, 13117 (76%) in stage III, and 12/18 (66%) in stage IV had PGLC-R 1 SD or more below control values. This decrease was not correlated with the decrease in the absolute number of lymphocytes or TEt or with the changes in TEa values.
400
GOUST
C
SI
ET
AL.
SII
Sill
SIV
FIG. 1. Percentages of PGLC-R in the control group (C), and in breast cancer patients tested 2-4 weeks after diagnosis in stages I (SI), II (SII), III (SIIK), and IV (SW). Bars indicate mean ? standard deviation.
Effect of Breast Cancer Sera on Rosette Formation by Normal Lymphocytes Sera from patients at various stages (whose TEt, TEa, and PGLC-R levels were determined on the day the serum was obtained) induced a small (15 2 4.8%) but significant (p < 0.05) decrease in TEt from normal donors, regardless of whether or not their own TEt were decreased (Table 4). TEa and PGLC-R remained unchanged. DISCUSSION
Previous studies of circulating T cells in breast cancer patients have shown decreases in the percentage of T cells (12, 13), associated with near normal or slightly increased percentages of B cells. However, as described in the present work lymphopenia is frequent in breast cancer and is more marked in patients at advanced stages of the disease. This is in agreement with the data of Glas et al. (6). The time between the beginning of the symptoms and surgery, limited to biopsy in stage IV, was frequently unknown; however, the patients in this study were first tested from 2 to 6 weeks after surgery, thus eliminating a possible cause of exogenous immunodepression. Hence, the lymphopenia at the time of testing could be considered as a first symptom of abnormal immune response. Therefore, it is necessary to take into account not only the percentages of various T-cell
T-CELL
MARKERS
IN
BREAST
TABLE CHANGES
IN
TEt, TEa,
AND WITH
NONSPECIFIC
SERA FROM
Value for
4 T
SUPPRESSION
11 BREAST
Normal control lymphocytes
70.5 k 3c
Lymphocytes from Patient 1 Normal lymphocytes incubated with serum from Patient 1
76.5 44
Lymphocytes from Patient 3 Normal lymphocytes incubated with serum from Patient 3
(6W 57 55 (78) 52 59 (83)
(PGLC-R)
AFTER
INCUBATION
PATIENTS
TEa (%)” 34.7 k 0.7 48.5 10.5 (30) 30 32 (94) 33 38 (111) 40 38.5
PGLC-R (%)” 30.0 * 0.5 33.5 (111) 24
630) 32.5 (32.5)
Lymphocytes from Patient 4 Normal lymphocytes incubated with serum from Patient 4
(t;)
(102)
(80)
Lymphocytes from Patient 5 Normal lymphocytes incubated with serum from Patient 5
77 56 (7%
45.5 35
30 24
(102)
(89
Lymphocytes from Patient 6 Normal lymphocytes incubated with serum from Patient 6
63 65 (92) 60 70.5 (109 47 62
49 34 (109 50 32.5 (104) 34 29
15 30 (109
(88)
(85)
Lymphocytes from Patient 7 Normal lymphocytes incubated with serum from Patient 7 Lymphocytes from Patient 8 Normal lymphocytes incubated with serum from Patient 8
74.5
CELLS
CANCER
TEt (%)”
Lymphocytes from Patient 2 Normal lymphocytes incubated with serum from Patient 2
401
CANCER
Lymphocytes from Patient 9 Normal lymphocytes incubated with serum from Patient 9
55 72 (102)
Lymphocytes from Patient 10 Normal lymphocytes incubated with serum from Patient 10
73 74 (104) 31.5 64
Lymphocytes from Patient 11 Normal lymphocytes incubated with serum from Patient 11
31 (lit) 40 32.5 (95) 25.5 35.5 (104)
19 31.5
23 39
(126) 10 29.5 (98.3) 10 39 (139 9 36.5
(121) -
n Percentage of total mononuclear cells. b Percentage of PGLC cells binding three or more lymphocytes. c Mean * SD. d Percentage of mean value for normal lymphocytes without incubation.
subsets but also millimeter cubid in this study the except stage IV,
their absolute numbers, expressed as the number of rosettes per of blood, as discussed previously by Kerman et al. (14). Indeed, percentage of TEt was near normal in almost all patient groups in contrast with the results of Keller et al. (12) and Whitehead et
402
GOUST
ET
AI..
al. (5); however, when the absolute number of TEt rosettes was calculated, a progressive decrease became obvious, which strictly paralleled the decrease in lymphocytes. One can thus consider that T cells, as a whole. remain in normal proportion within the circulation, although the more advanced the disease is at the time of diagnosis, the more their total numbers are decreased. In this study we found a decrease of TEt percentages in only a few patients, indicating that the receptors for sheep erythrocytes were not masked on the cells from most patients. This may be due to our extensive washing of the cells before rosette tests were performed, removing an inhibitor (5), which might be antigen-antibody complexes causing steric hindrance. Such an inhibitor, if present, apparently did not affect active rosette-forming cells (TEa) in this study nor the population of PGLC rosette-forming cells. The increased percentage of TEa in stage I and, to a lesser extent, in stage II breast cancer patients suggests a possible prognostic significance. These cells have been shown to proliferate actively in the peripheral blood (15) and be involved in T-cell-mediated cytotoxicity (16), and their role in immunosurveillance in cancer patients has been emphasized (8, 9). Their increase in stage I may indicate an ongoing immune response against the tumor. However, further observation is needed to determine whether patients with high TEa in stage I may have a relatively longer disease-free interval after surgery, whereas those with low TEa would require immunotherapy. On the other hand, it has been reported in one clinical study (17) that the percentages of relapse 5 years after diagnosis were respectively 23% for patients without lymph node involvement. 49% when one to three nodes were involved, and 81% when four or more nodes were involved. It is noteworthy that the percentage of patients in the present study with absolute numbers of TEa 1 SD or more below normal values was, at each stage, very close to the 5-year recurrence rate previously reported. Longitudinal studies have recently been initiated in this laboratory to ascertain whether stage I and II patients with high TEa levels have a normal life span whereas those with low TEa may develop metastases within a few years after surgical removal of the primary tumor. The binding of peripheral blood T cells to B-LCL occurs through receptors which have not been identified yet, but it apparently does not involve Fc or E-rosette receptors. Our studies of this phenomenon (J. M. Goust, manuscript in preparation) show that only two-thirds of normal peripheral blood T cells and thymocytes are able to bind to LCL. It is, therefore, possible that these T cells may belong to a specific subset, possibly involved in T-B cell interactions, as suggested by Jondal er al. (10). It might have been more logical in this study to express the data as percentage of MNC bound to LCL. However, in patients with a low percentage of PGLC-R, the percentage of MNC bound to B-LCL was consistently lower than the value observed in normal controls, and it is technically easier to count the percentage of rosetted B-LCL than to determine the percentage of free and bound MNC. The reduction in the subset of T cells binding to this cell line in advanced disease may reflect either the selective masking of a T-cell receptor or the disappearance of a specific subset bearing the receptor, since it is not correlated with decreases in other subsets of T cells. In a previous study (11). we found that 70% of multiple sclerosis patients had both decreased PGLC-R and
T-CELL
MARKERS
IN
BREAST
CANCER
403
increased immune complexes, assessed by the Clq-binding technique. A similar inverse correlation could exist in breast cancer. Indeed, Rossen et al. (18) have reported increased Clq binding in 74% of breast cancer patients, with the highest values obtained in patients who had active disease. It is possible, therefore, that the disappearance of PGLC rosette-forming T cells may allow the production of antibodies to an etiologic agent(s), followed by immune complex formation, which in turn would lead to further impairment of cell-mediated immune responses. ACKNOWLEDGMENTS The help of M. Keels, R. N., in collecting blood samples and clinical information is gratefully acknowledged. The reliable and skillful technical assistance of Gena Ciabattari has been invaluable for this work. We thank Charles L. Smith for excellent editorial assistance.
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Catalona, W. J., Sample, W. F., and Chretien, B., Cancer 31, 65-71 (1973). Dellon, A. L. Potvin, C., and Chretien, P. B. Cancer 35, 687-694 (1975). Stefani S. S., Kerman, R., and Abbate, J., Amer. J. Roentgenol. 126, 880-886 (1976). Stefani, S. S., Kerman, R., and Abbate, J., Cancer 37, 2792-2796 (1976). Whitehead, R. H., Roberts, G. P., Thatcher, J., Teasdale, C., and Hughes, L. E., J. Nut. Cancer Inst. 58, 1573-1576 (1977). Glas, U., Wasserman, J., Blomgren, H., and DeSchryver, A.,lnr. J. Radiat. Oncol. Biol. Phys. 1, 189- 195 (1976). Stjernsward, J., Jondal, M., Vanky, F.. Wigzell, H., and Sealy, R., Lancet 1, 1352-1365 (1972). Wybran, J., and Fudenberg, H. H., In “Clinical Tumor Immunology” (J. Wybran and M. Staquet, Eds.), pp. 31-40, Pergamon, London (1976). Wybran, J., and Fudenberg, H. H., J. Clin. Invest. 52, 1026-1031 (1973). Jondal, M., Klein, M., and Yeyenoff, E., Stand. J. Immunol. 4, 259-266 (1975). Goust, J. M., Carnes, J. E., Chenais, F., Hames, C. G., Fudenberg, H. H., and Hogan, E. L., Neurology 28, 421-425 (1978). Keller, S. E., Ioachim, H. L., Pearse, T., and Siletti, D. M., Amer. J. C/in. Pathol. 65,445-449 (1976). Nemoto, T., Han, T., and Minowada, J., J. Nut. Cancer Inst. 53, 64-645 (1974). Kerman, R. H., Smith, R., Stefani, S. S., and Ezdinli, Z., Cancer Res. 36, 3274-3278 (19761. DeBruyere, M., Lachapelle, J. M., and Anguita, T., Int. Arch. Allergy Appl. Immunol. 54,50-57 (1977). Wybran, J., Hellstrom, I., Hellstrom, K. E., and Fudenberg, H. H., Int. J. Cancer 13, 515-521 (1974). Fisher, B., Black. N. H., and Brose, I. D. J., Cancer 2, 1071-1080 (1969). Rossen, R. D.. Reisberg, M. A., Hersh, E. M., and Gutterman, J. V., J. Nat. Cancer Inst. 58, 1205-1215 (1977).