Int. J. Cancer: 19, 487-497 (1977)

RELATIONSHIP OF HUMAN NATURAL LYMPHOCYTE-MEDIATED CYTOTOXICITY TO CYTOTOXICITY OF BREAST-CANCERDERIVED TARGET CELLS Grace B. CANNON l, Guy D. BONNARD 2, Julie DJEUI, William H. WEST and Ronald B. HERBERMAN Department of Immunology, Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland 20795: and Laboratory of Imrnunodiagnosis, National Cancer Institute, National Institutes of Health, Public Health Service, US Department of Health, Education and Welfare, Bethesda, Maryland 20014, USA

Mononuclear cells f r o m 115 individuals were tested in a 4-h chromium release assay against t w o breastcancer-derived cell lines, G11 and MCF-7, and a myeloid line, K-562, shown previously t o be sensitive t o natural cytotoxicity. These data were analyzed In a manner designed t o detect hyperreactivity against the breast cell lines relative t o the level o f reactivity against K-562. A high proportion o f breast cancer patients were found t o be relatively hyperreactive against G11 (12/18 o r 67%) and against MCF-7 (loll8 o r 56%). Fibroadenoma patients were very similar t o the normal females, w i t h O / l l hyperreactive t o G l 1 and 1/11 (9y0) t o MCF-7. However, several normal males (7/17 o r 41%) were hyperreactive t o G11 but n o t t o MCF-7(2/17 o r 12%). Colon cancer and lung cancer patients were also more hyperreactive t o G11,4/8 o r 50% and 416 o r 67%, respectively, than they were t o MCF-7, l / 8 o r 13% and 1/6 o r 17%, respectively. Only fibrocystic patients resembled the breast cancer patients, w i t h some but not as many individuals being hyperreactive t o G l 1 (3/8 o r 38%) and t o MCF-7 (2/8 o r 25%). W i t h another group o f individuals reproducibility of the method was demonstrated, w i t h only 1/14 or 7% o f normal females and 12/17 o r 70% o f breast cancer patients being hyperreactive t o G11. Thus, natural cytotoxicity toward K-562 can be related t o breast cancer-associated cytotoxicity toward MCF-7 i n a way that distinguishes a majority o f brest cancer patients specifically f r o m other groups o f individuals.

Lymphocytes from cancer patients were initially reported to be specifically cytotoxic for cultured cells derived from tumors of the same type (Hellstrom et al., 1971; Fossati et al., 1971; O’Toole et al., 1972). However, in the past several years many investigators have found that lymphocytes from normal donors are often as cytotoxic as from cancer patients, or more so, for a variety of tumorderived cell lines (Oldham et al., 1973; Takasugi et al., 1973; Rosenberg et al., 1974; Peter et al., 1975; Kay and Sinkovics, 1974; Oldham et al., 1975). In a recent discussion of this problem, it was suggested that natural reactivity may co-exist with disease-related cytotoxic reactivity, and that the specificities or effector cells of each type of cytotoxicity may be different (Herberman and Oldham, 1975). Consistent with this, Jondal and Pross (1975) reported that natural cytotoxicity

could be eliminated by removal of complement receptor-bearing lymphocytes and that specific reactivity against Epstein Barr virus (EBV)-associated antigens could then be detected (Svedymyr and Jondal, 1975). Subsequently West et al. (1976) found that Fc receptor-bearing lymphocytes were responsible for natural cytotoxicity, but again this provided a basis for separation of different types of effectors cells. The present study was based on the possibility that breast cancer patients have both natural cytotoxic reactivity and breast-tumor-related cytotoxic reactivity and that this might lead to disproportionately more cytotoxicity of cultured cells derived from breast cancer than of cultured cells from other sources. Lymphocytes from breast cancer patients and from controls were, therefore, tested at the same time against two cell lines derived from breast cancer and against K-562, a myeloid cell line which has been found to be particularly sensitive to natural cytotoxicity (Jondal and Pross, 1975). This study was performed with a short-term 51Cr release cytotoxicity assay which allows more accurate and rapid quantitation than the visual microcytotoxicity assay used in many previous studies. The studies of Levin et al. (1975) had indicated that some monolayer cell lines were sensitive to short-term cytotoxicity. MATERIAL AND METHODS

Source of blood specimens

Heparinized blood specimens were obtained from patients with breast cancer and other diseases from the Medical University of South Carolina and from the George Washington University Hospital. With the specimens from South Carolina, the lymphocytes were separated there by the Ficoll-Hypaque Received: November I , 1976 and in revised form December 20, 1976.

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CANNON ET AL.

procedure (see below) and sent to our laboratory on wet ice. As controls for this extra handling, lymphocytes from normal donors were obtained similarly from hospital personnel at the Medical University of South Carolina. Local normal donors included Litton Bionetics' clerical and laboratory personnel.

counter. Percentage cytotoxicity was calculated according to the following formula:

Isolation of mononuclear cells

The baseline or autologous control contained unlabelled target cells in place of, and at the same concentration as lymphocytes.

Mononuclear cells were separated from heparinized whole blood by centrifugation on a FicollIsopaque gradient for 30 min at 400 g, according to the method of Boyum (1968).

% Cytotoxicity = Counts per minute (CPM) in supernatant of experimental culture -CPM in supernatant of baseline control x 100. Total CPM initially incorporated into cells

Statistical analysis

The data were analyzed in the manner suggested by McCoy et al. (1976) for the leukocyte migration assay. The reactivity of the cancer patients was Two breast-cancer-derived cell lines, G 11 and evaluated after a large population of normal indiMCF-7, and a myeloid cell line, K-562, were used viduals was examined. Only the first tests of each in the tests. G11, a clone of the cell line HBT-3 of these normals as well as of cancer patients were established from a breast tumor by Bassin et al. used so that the study would not be biased by (1973), was obtained from Dr. E. Plata. MCF-7 several tests from one individual and a few from was obtained from Dr. H. D. Soule who had estab- other individuals. Later, subsequent tests of normal lished it (Soule et al., 1973). Both these lines grew individuals and cancer patients were studied and as monolayers. K-562 was established by Lozzio used to examine reproducibility. In examining the and Lozzio (1973) and was initially obtained from normal data, the reactivity of some normal males Dr. George Klein. It grew in suspension culture. differed from that of normal females. Consequently All cell lines were grown in RPMI 1640 and 20% the results of the tests with normal males were filtered, heat-inactivated fetal bovine serum. All placed in a separate group. A cut-off based on the cell lines with the exception of K-562 were main- reactivity of the first tests of normal females was tained free of mycoplasma and checked frequently set up to evaluate the reactivity of the first tests of for it by the method of Fogh and Fogh (1969). cancer patients. The cut-off was set at approximately We found K-562 to have mycoplasma upon arrival the upper tenth percentile of reactivity of normal in our laboratory and maintained it on gentamycin females and values above this cut-off were considered (5 mg/100 ml medium) to keep the level down. to be in the high range or false positive tests. The Subsequently, another K-562 culture free of myco- proportion of individuals in the other test groups plasma was obtained from Drs. C. B. Lozzio and falling into this abnormal range was determined. B. B. Lozzio. The monolayers were trypsinized with The Wilcoxon ranking test was done on certain a trypsin-EDTA solution (0.5 g trypsin and 0.2 g individual test results of the various groups to show EDTA per liter of Modified Puck's Saline A) and differences or similarities among the cell lines or suspended in media just prior to the test. groups (Snedecor and Cochran, 1975). In these studies, most of the individuals were Cytotoxicity test tested at the same time, against each of the three This procedure has been described elsewhere in cell lines. This allowed us to analyze the cytodetail (Rosenberg et al., 1974). The target cells toxicity data dependently, i.e., to compare directly were labelled with 0.15 mCi (1 mCi/ml) W r for the level of cytotoxicity of each individual against 45 min in a 37" C water bath with shaking. After one cell line with the level against another line. The relationship of the cytotoxicity of individuals washing with hepes buffered RPMI 1640 containing 10% fetal bovine serum and gentamycin to the various lines was evaluated by examining the (0.05 mg/l ml), 1 x lo8 lymphocytes were added to simultaneous test results of the lymphocytes against 1 x lo4 target cells, a ratio of 100:1, in triplicate two cell lines on a two-dimensional graph. By plastic screw-top tubes in a total volume of 1.05 ml. comparing, on a population basis, test results of After 4 h incubation at 37" C in a 5 % CO, incubator normal females with those of the various test on a rocking platform (six cycles per minute), groups, differences among the groups were obvious. 1 ml of cold medium was added and the tubes A diagonal cut-off based on the pattern of normal were centrifuged at approximately 1,000 g for 10 min. female reactivity was set at approximately the top Each supernatant was poured into a counting tube tenth percentile of normal female reactivity (see and counted for 4 to 10 min in an automatic gamma above). The proportion of individuals in the other History and preparation of target cells

NATURAL AND BREAST-CANCER-RELATED CYTOTOXICITY

test groups was compared to this low level of false positives among the normal females. Correlation coefficients were calculated on data from these simultaneous test results according t o the method of Snedecor and Cochran (1975). Then the various correlation coefficients (r) from the various cell line combinations were compared. RESULTS

Cytotoxicity for three cell lines analyzed independently

K-562. The results from the first 115 individuals tested were divided into seven different categories : normal females, normal males, and patients with breast cancer, fibrocystic disease of the breast, fibroadenomas, lung cancer, and colon cancer. The levels of cell-mediated cytotoxicity against K-562 were remarkably similar among the different groups on a population basis, but values varied among individuals from 5% to 60% (Fig. 1). As can be seen in Table I, the mean and median per-

SCATTERGRAM OF C Y T O T O X I C I T Y TO THREE D I F F E R E N T CELL L I N E S

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FIGURE 1 Cell-mediated cytotoxicity of normal females and males, breast cancer patients, benign breast disease (fibrocystic and fibroadenoma) patients, and lung and colon cancer patients against a myeloid cell line, K-562, and two breast-cancer-derivedcell lines, G11 and MCF-7. Each point represents the results of the first test of each individual.

489

490

CANNON ET AL.

centage cytotoxicities of the various groups were quite similar. When a Wilcoxon ranking test was done on the two most disparate groups (colon and lung cancer), they were not quite significantly different (p = 0.06). If a cut-off for high reactivity was set based on the upper eleventh percentile of normal female reactivity, a similar proportion of individuals in each group except the colon cancer group had high reactivity. A larger proportion of colon cancer patients (5/8) had high reactivity. G I ] . The individual cytotoxicity values to this line ranged from 0% to 45% (Fig. 1) but on a population basis the groups were not significantly different from each other by the Wilcoxon test. For example, a comparison between the levels of cytotoxicity for normal females and breast cancer

I C A i l I R G R A M OF S I H U L T A N t U U S

reactivity, was slightly more than that of other cancer patients (28% versus 14%). Cytotoxicity for three cell lines analyzed dependently Figure 1 shows the reactivity of individuals in various groups against each of three different cell lines but does not indicate the pattern of reactivity of each specimen against the three cell lines. One cannot determine if a low reactor to one line was also a low reactor to another line. For this reason we have plotted the cytotoxicity values for each individual against two different cell lines on a two-dimensional graph. In this way, the relative reactivity against both lines can be evaluated. Based on the distribution of the reactivity of the normal females in the scattergram, a diagonal cut-off

R L A C T I Y I T I T O G I 1 AN0 1 5 6 2

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Cytotoxicity of individuals against two cell lines ((311 and K-562) tested at the same time. Listed below title of each graph is mean percentage cytotoxicity to each line, the proportion of individuals with hyperreactivity to G11-K-562 according to the diagonal cut-off mentioned in the text, and the correlation coefficient, r. An asterisk indicated that the correlation was significant at pG0.05. Each point represents the results of the first test of an individual. In the upper right-hand graph w stands for males with lung cancer, o stands for females with colon cancer and 0 for males with colon cancer.

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patients gave p = 0.06. If a cut-off for high reactivity was set at the upper eleventh percentile of normal female reactivity, then the proportion of high reactors in the various groups did vary, with lung, breast, and colon cancers being the most reactive at frequencies of 67 %, 61 % and 50%, respectively (Table I). This increased proportion of high reactivity in test groups was also reflected by the higher means and medians of these groups. MCF-7. The cytotoxicity values for breast cancer patients did not differ from those of the normal females by the Wilcoxon test, with p = 0.09. However, the proportion of breast cancer patients with high reactivity to this line, based on the upper eleventh percentile cut-off for normal female

has been set to examine hyperreactivity against a breast-cancer-derived cell line relative to that against K-562. As before, a cut-off was selected which would separate most normal females from the upper 11 %. K-562-GII. The scattergrams in Figure 2 show that the relative levels of cytotoxicity of many of the breast cancer patients against G I 1 and K-562 were quite different from the pattern of most normal females. The cytotoxic reactivities of normal females against the two lines were significantly correlated. In contrast, with breast cancer patients there was a poor correlation in activities against the two lines [correlation coefficient (r) = +0.12]. Many of the breast cancer patients had cytotoxic activity

NATURAL AND BREAST-CANCER-RELATED CYTOTOXICITY

491

females. The correlation coefficients (+0.79 for lung and $0.65 for colon) were high but not significant at p = 0.05. Thus the pattern of reactivity revealed by this method did set most normal females and fibroadenoma patients apart from the majority of breast cancer patients. However, the pattern of G11 hyperreactivity was not specific for breast cancer, and a similar pattern was seen in patients with lung cancer, colon cancer and fibrocystic disease, and with normal males. K-562 - MCF-7. When the cytotoxicity values of simultaneous tests against K-562 and MCF-7 were plotted graphically (Fig. 3), a more clear-cut difference between the patterns of patients and of normal donors was seen. The only group to show a significantly positive correlation coefficient between reactions against the two lines was that comprising

against G11 irrespective of their concomitant natural reactivity against K-562. When the 11 % normal female cut-off was used, 67% (12/18) of the breast cancer patients were hyperreactive against G11 (Table I). The pattern of reactivity of the fibroadenoma patients was similar to that of normal females in that their cytotoxicity values toward the two lines were significantly correlated (r = +0.72). When the diagonal cut-off was used, none of them were hyperreactive to G11. The reactivities against the two lines by fibrocystic patients appeared to be correlated (r = +0.63), but this was not significant. With the diagonal cut-off, three of eight patients fell into the GI 1 hyperreactive area. The pattern of the reactivity of normal males appeared to be very different from that of normal females. The cytotoxic levels for the two lines were

S C A T T E R G R A M O F S I M U L T A N C O U S R E A C l i Y I T V 10 M C F - 7 AM0 1 5 6 2

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Identical to Figure 2, except for cell lines tested. Here, cytotoxicity against MCF-7 and K-562 of each individual is plotted.

FlBROAOENOMA PATIENTS

FIBROCYSTIC PATICUTS MCF-7: 81

BREAST CANCER P U T I C N l S MCF-7: > O X

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not significantly correlated (r = $0.28). With the diagonal cut-off, 7/17 (41 %) of the normal males fell into the G11 hyperreactive part of the graph. Thus, their reactivity showed a similarity to that of the breast cancer patients. If the male reactivity was analyzed according to source, 4 out of 10 South Carolina male hospital workers were reactive. Three out of 7 Bionetics male employees were positive, indicating that the two groups of males were similarly reactive. Due to the elevated reactivity of the normal males by this analysis, the other cancer patients were analyzed with respect to sex. Four of 6 lung cancer patients (all males) and 3 of 5 colon cancer patients (males) were reactive, as was 1 of 3 colon cancer

10

20

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50

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the fibroadenoma patients. However, the fibrocystic, colon, and lung cancer patients had fairly high correlation coefficients (f0.62, $0.63, $0.67). With the breast cancer patients, there was no correlation between reactivity against MCF-7 versus that against K-562 (r = +0.05). The uniqueness of reactivity of breast cancer patients against MCF-7 is clearly demonstrated in Figure 3. As seen with G11, the proportion of breast cancer patients hyperreactive to MCF-7 was much higher (56 %) than the proportion of normal females demonstrating similar reactivity (11 %). In contrast to studies with G11, normal males and patients with colon and lung cancer failed to demonstrate disproportionate reactivity against MCF-7 and appeared

492

CANNON ET AL. SCATTERGRAM OF SIMULTANEOUS

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essentially the same as normal females. Nine percent of the fibroadenoma patients and 25% of the fibrocystic patients were relatively hyperreactive against X-562 and MCF-7. Thus, simultaneous analysis of reactivity against K-562 and MCF-7 and identification of individuals demonstrating disproportionate reactivity to the breast-cancer-derived line permitted good discrimination between the reactivity of breast cancer patients and other test groups. G I 1 - MCF-7. It was also important to evaluate the simultaneous reactivity of these same individuals

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FIGURE 5 Confirmation of the test method with simultaneous cytotoxicity values of another group of normal females and breast cancer patients. Mean cytotoxicity, proportion reactive, and correlation coefficient (r) are listed below the titles. In the four graphs in the right-hand part of the figure, repeat tests of an individual are designated by numbers. For the first normal female, repeat tests were 1 1/2 months, 1 week, 1 week, 1 week, 1 1/2 months, 2 months and 3 weeks apart. For another normal female repeat tests were 2 weeks, 3 months and 3 months apart and for the other normal female, 0, tests were 4 months, 2 1/2 months and 2 months apart. Breast cancer patients' tests were the following times apart: 0 = 9 months, 0 = 7 months, A = 1 week, 1 1/2 months, x = 2 weeks. Normal males' tests were the following times apart; 0 = 2 weeks, 0 = 3 weeks, = 3 weeks, between tests l + 2 and 2 weeks between tests 2 and 3, A = 3 weeks, X = I week.

against GI1 and MCF-7 (Fig. 4), to determine whether high reactors to one line were the same as high reactors to the other line. Against these lines the reactivities of normal females, normal males, breast cancer patients and fibroadenoma patients were significantly correlated. The lack of statistically significant correlation of the reactivities of the fibrocystic, lung cancer and colon cancer patients may be related to the low numbers of individuals in these categories. Using both of the individual hyperreactivity cutoffs for these two cell lines in Figure 4, one can then

NATURAL AND BREAST-CANCER-RELATED CYTOTOXICITY

easily see whether each donor had hyperreactivity against either line or against both. Only 3 individuals (1 normal female, 1 breast cancer patient and 1 fibrocystic patient) had high reactivity only to MCF-7 and not to G11. However, 18 individuals had high reactivity to G11 and not to MCF-7: 1 normal female, 3 normal males, 2 lung cancer patients, 2 colon cancer patients, 7 breast cancer patients, 2 fibrocystic patients, and 1 fibroadenoma patient. Only 10 individuals had high reactivity to both breast-cancer-derived cell lines : 2 normal females, 2 normal males, 1 lung cancer patient, 1 colon cancer patient, 4 breast cancer patients and none of the benign breast disease patients. This correlation graph does not show which of the breast cancer patients had high reactivity to the breast-cancer-derived cell lines relative to that against K-562. Eight of the breast cancer patients were positive against both MCF-7 and G11 relative to K-562. Three of the breast cancer patients were negative with the K-562-MCF-7 cut-off, but were positive with the K562-Gl1 cut-off. Only one breast cancer patient was positive with the K-562MCF-7 cut-off and negative with the K-562-G11 cut-off. Confirmation of the C11-K-562 reaction with another group of individuals

During the course of the testing reported above, on some test days, individuals were tested against only two cell lines, K-562 and G11. The results of this data have been analyzed to show the reproducibility of the dependent method of analysis. These are individuals similar to, but not the same as, those in the first group whose results are summarized above. Again, the levels of reactivity of breast cancer patients were compared to those of normal females (Fig. 5). The diagonal cut-off was set so that only one female (7 %) would be considered to have relatively high activity against G l l . Using this cut-off, 70% (12117) of the breast cancer patients were hyperreactive against G1 1. Therefore, although the absolute levels of cytotoxicity against each line independently tended to be lower than in the previously analyzed tests, the pattern of results was quite similar. Again, the levels of cytotoxicity of normal females against the two cell lines were significantly correlated, whereas those of the breast cancer patients were not. Thus, the test method seems to apply to different sets of data collected from two different groups of normal females and breast cancer patients. Up to this point, this report has dealt with results of the first test performed with each individual. Many of the patients and normal donors were tested repeatedly and, in Figure 5, some of these results

493

are shown, Two normal females varied within the normal range except for one test each. Another normal female had two of four tests within the G11 hyperreactive range. Two breast cancer patients were hyperreactive to G11 on two consecutive tests. However, three other breast cancer patients who were initially hyperreactive t o G11 had their repeat tests in the normal range. One normal male was repeatedly hyperreactive and another was repeatedly in the normal range. Three other males fluctuated from the hyperreactive to the normal range. From this and other data we have found that most (90%) normal females tested repeatedly tend to remain in the normal range. Normal males and breast cancer patients tend t o fluctuate between the normal and hyperreactive ranges. Clinical information on breast cancer patients in the study

Clinical information on the breast cancer patients in this study has been analyzed to determine whether any parameters correlated with hyperreactivity to the breast-cancer-derived cell lines (Table 11). The age of the patients seems unrelated in that some patients in all age groups were hyperreactive to one of the two lines. The stage of the patients was either I (no lymph nodes involved) or I1 (one o r more lymph nodes involved). None of the patients were in stage I11 (metastatic disease present at time of diagnosis). At least 50 % of the patients were hyperreactive regardless of stage, indicating that stage was not an important factor in determining whether they were reactive. Only 4 of the 35 breast cancer patients had metastatic disease at the time of the test. Three of these 4 were reactive, a similar level of reactivity to that seen in patients who had no evidence of disease at the time of the test (17125) and in the preoperative patients (416). Thus, the presence of tumor was not an inhibitor of the hyperreactivity. Preoperative patients as well as postoperative patients even several years after mastectomy were hyperreactive. There were low periods of reactivity at 2 weeks to 1 month post operation and at 6 months to 1 year. However, within the first 2-week period post operation, 75 % of the patients were reactive. Eleven of the breast cancer patients in the study were treated with radiation. A reasonable percentage (>SO%) of the tests were positive at all periods following radiation except during the first month. Here, three of four patients were non-reactive indicating that the radiation treatment may have influenced the test. Consequently the four patients who were tested within 1 month of radiation were eliminated from the study and were not included in any of the previous analyses.

494

CANNON ET AL. TABLE I1 CLINICAL INFORMATION OF BREAST CANACER PATIENTS TESTED WITH K-562,G11, AND MCF-7

Proportion of individuals hyperreactive to

Age of patient

< 39

40-44

45-49

50-54

55-59

60-64

Total

Stage I1

Stage I

MCF-7

70->70

65-69

6/10 8/10 518

G11 (1) GI1 (2)

Current disease status Post-operative patients

Pre-operative patients Local disease only

No evidence of disease

Distant metastatic disease mesent

213 213 213

7/14 9/14 811 I

Ill

MCF-7 G I 1 (1) GI1 (2)

Ill 213

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MCF-7 GI1 (1) GI1 (2)

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213

112

GI I (1) refers to the same group of patients and normal individuals who were tested with MCF-7 and K-562.Thus, the denominator in GI1 (1)is the same as that in MCF-7. GI1 ( 2 ) refers to another group of patients an! normal individuals who were only tested with K-562 and G1 I and not with,,MCF-7. These were analysed in the section of ‘‘ Results ” entitled Confirmation of the (311-K-562 reaction with another group of individuals.

DISCUSSION

As previously discussed (Herberman and Oldham, 1975), many investigators have suggested that the

occurrence of natural cell-mediated cytotoxic reactivity of normal individuals as well as of cancer patients seriously interferes with the practical application of cytotoxicity assays to the study of selective cell-mediated immunity of cancer patients against cultured cells derived from the same type of tumor. In our experience, and in many other laboratories, qualitatively positive tests, i.e., cytotoxicity significantly above the baseline controls, may occur with the same frequency among normal individuals and patients with other types of cancer.

In the present study, we have attempted to quantitate the levels of cytotoxicity of different individuals, to determine whether patients with breast cancer have higher levels of cytotoxicity against some cell lines than do other patients or normal donors. The 51Crrelease assay was found to be considerably more appropriate and convenient for this approach. The main limitation imposed by this assay is the need for cell lines which are sufficiently sensitive to rapid cytotoxicity. The K-562 cell line, as previously reported by Jondal and Pross (1975), has been found to be particularly sensitive to natural cell-mediated cytotoxicity and was considerably more suitable for these studies than the F265 or Raji cell lines which we previously used

NATURAL AND BREAST-CANCER-RELATED CYTOTOXICITY

(Rosenberg et al., 1974; McCoy et al., 1973). The G11 cell line, as previously reported by Levin et al. (1975), and the MCF-7 cell line, despite their being monolayer lines, were also sufficiently sensitive to rapid cytotoxicity. All of the data reported here were from tests with 1ymphocyte:target cell ratios of 1OO:l. Although this does not yield as much quantitative information as complete dose response curves, this procedure was more practical for testing of large numbers of specimens and did provide clear indications of the relative trends of cytotoxic reactivity among individuals. In the instances when several 1ymphocyte:target cell ratios were tested, the curves supported the relative differences indicated by the 1OO:l ratio. When the levels of cytotoxicity of the various groups were compared by a ranking method on the populations, no significant differences were seen. However, it was possible to select a cut-off for the normal female population to identify a higher proportion of breast cancer patients with high reactivity against the breast-cancer-derived cell lines. In contrast to the 11 % set for normal females, 61 % of the breast cancer patients had high reactivity against G11 and 28% of them had high reactivity against MCF-7. Although the tests with MCF-7 appeared to be less sensitive for discriminating breast cancer patient reactivity, they were also more specific. With G11, patients with a variety of other types of cancer had about the same incidence of high reactivity as did the breast cancer patients. Although the results of tests against each line, when analyzed independently, gave some suggestion of breast-cancer-related cytotoxicity, this trend was seen considerably more clearly when the results of each specimen against MCF-7 and K-562 were compared. It was possible to set a discriminant or cut-off to indicate relative hyperreactivity against the breast-cancer-derived line. By this analysis, the majority of breast cancer patients tested had relatively high activity against MCF-7, whereas only about 10% of most of the other groups behaved similarly. Only the fibrocystic disease patients had an intermediate incidence of hyperreactivity. This is of potential interest since there appears to be an increased incidence of breast cancer among such patients as compared t o patients with fibroadenomas or to females without benign breast disease (Donnelly et al., 1975). It also should be noted that patients with fibrocystic disease have an increased incidence o f cell-mediated reactions to extracts of MCF-7 in the leukocyte migration inhibition assay (Cannon et ul., 1977). When the results of tests against GI1 and K-562 were analyzed together, there was no improvement in discrimination between breast cancer patients and other groups, compared to the analysis of

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hyperreactivity to G11 alone. In fact, the relative comparison introduced a new problem, apparent hyperreactivity of normal males against G11. It is not clear why this pattern of reactivity differs from that of normal females. However, it raises the question of hormonal influences on the pattern of cytotoxicity o r on the antigenic specificities recognized. From the results, it can be concluded that MCF-7 is a more satisfactory cell line, than GI1 for the study of breast-cancer-associated cytotoxicity. In the last few years, MCF-7 has been studied extensively and has been found to possess a variety of markers or characteristics of breast tumor cells: it produces estrogen receptors and alphalactalbumin (Lippman and Bolan, 1975) and upon inoculation into nude mice it produces tumors with the characteristic appearance of breast adenocarcinomas (Giovanella, personal communication). It has also been found to be a good source of antigen for leukocyte migration inhibition studies of cell-mediated immunity of breast cancer patients (McCoy et al., 1976). In contrast, G11 lacks the biochemical markers (Lippman and Bolan, 1975), and Nelson-Rees et al. (1974) have raised the question of HeLa cell contamination in this line. G11 does produce in nude mice tumors which are adenocarcinomas, consistent with breast origin and clearly distinguishable from HeLa related tumors which are undifferentiated or epidermoid carcinomas (Fogh and Hajdu, unpublished observations), but it remains possible that G11 has undergone some genetic recombination with HeLa cells and has lost some breast-cancerassociated traits, including antigens. The present study indicates that a cytotoxicity assay can be used to detect patterns of cell-mediated reactions which are particularly characteristic of breast cancer patients, and this tends to support previous conclusions that many breast cancer patients have cells which are specifically cytotoxic to breast cancer cell lines (Hellstrom et al., 1971 ; Fossati et al., 1972, Heppner et al., 1973). The need to compare levels of cytotoxicity of breast cancer patients against MCF-7 with levels against K-562 suggests that the lymphocytes of breast cancer patients have two possible mechanisms for directly damaging cultured target cells : specific cytotoxicity against breast cancer-associated antigens and natural cytotoxicity of the same kind as that found in other patients and in normal donors. These data provide a considerable impetus to attempts to separate the possibly different cell subpopulations responsible for each type of cytotoxicity, along the lines utilized by Jondal and Pross (1975), and also to efforts to devise other means to more clearly study each type of reactivity separately and to determine its specificity in detail.

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ACKNOWLEDGEMENTS

The authors are indebted to Mrs. Marsa Aultman, Mr. Lenwood Keyes, Miss Elizabeth Read and Miss Katherine Pffifner for performing the assays, to Mrs. Diana Berard and Mrs. Nancy Merkel for tissue culture of the cells and mycoplasma monitor-

ing of the cells, and to Mrs. Marian Keels for isolation of the mononuclear cells. This study was supported in part by Public Health Service contract NIH-NO-CB-63975 from the National Cancer Institute, National Institutes of Health, Department of Health, Education, and Welfare, Bethesda, Maryland 20014.

RELATIONS ENTRE LA CYTOTOXICITE NATURELLE A MEDIATION CHEZ L'HOMME ET LA CYTOTOXICITE CONTRE LES CELLULES-CIBLES

LYMPHOCYTAIRE DE CANCER DU SEIN

Des cellules mononucleaires de 115 sujets ont 6t6 testies, dans une lpreuve de liberation du chrome d'une durCe de 4 h. contre deux lignees de cellules de cancer mammaire, G11 e t MCF-7, e t une lignee my6lolde, K-562, que I'on savait sensible la cytotoxicite naturelle. Les donnees obtenues ont 6t6 analys6es de fason P comparer I'hyperr6activitb contre les lign6es de cellules de turneur mammaire e t la reactivit6 contre l a lign4e K-562. O n a constat6 qu'une forte proportion des malades atteintes de cancer du sein 6taient relativernent hyperreactives aux cellules G l l (12/18, soit 67%) e t aux MCF-7 (lO/l8, soit 56%). Les patientes atteintes defibro-adhorne se rapprochaient nettement des fernrnes normales avec O / l l cas d'hyperreaction aux G11 e t 1/11 (9%) aux MCF-7. Toutefois, plusieurs homrnes normaux (7/17, soit 41%) pr6sentaient une hyperreaction aux G11 mais pas aux MCF-7 (2/17, soit 12%). Les malades atteints de cancer du c6lon ou du poumon dtaient egalement plus hyperreactlfs aux G11 (4/8 (50%) e t 4/6 (67%), respectivement) qu'aux MCF-7 (l/8 (13%) e t 1/6 (17y0), respectivement). Seules les personnes atteintes de maladies fibrokystlques se rapprochaient des cas de cancer du sein, avec cependant moins de car d'hyperrhaction aux G11 (3/8, soit 38%) e t aux MCF-7 (2/8, soit 25%). La reproductibilite de la methode a Ct6 d6montrCe avec un autre groupe, oh 7% des fernrnes norrnales (1/14) e t 70% des malades atteintes de cancer du sein (12/17) etaient hyperr6actives aux G11. On peut donc etablir un rapprochement entre la cytotoxicitC naturelle enverr ler cellules K-562 e t l a cytotoxicitk enverr les MCF-7, qui est associCe au cancer du sein, de fason P differencier specifiquernent la rnajorite des cas de cancer du sein d'autres groupes de personnes.

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