Journal of Surgical Oncology 11:217-225 (1979)
The Effect of Anticancer Therapy on Peripheral Blood T and B Lymphocyte Counts and Function .......................................................................................... .......................................................................................... T. L. HADFIELD, PhD, S. MARCUS,
PhD, and C.
R. SMART, MD
Patients categorized according t o tumor type were compared to a control nontumor population. Comparison of relative T cell values among the groups showed no significant differences; however, when absolute numbers of T cells/ mm3 were compared, all cancer patients, whether from treated or untreated groups, had significantly depressed T cell values. No significant differences were observed in the relative or absolute numbers of B cells. Comparison of the total lymphocyte response t o PHA showed no significant differences among the various cancer groups; however, response in all cancer groups whether from treated or untreated patients, was depressed by comparison to the control group. Patients categorized according to the type of treatment received showed significant depression in the white blood cell count, lymphocyte count, relative and absolute T cell counts and t h e absolute B cell count in the postsurgery, postadjunctive therapy group. T h e pretherapy group also showed significant depression in the absolute number of T cells/mm3 when compared t o t h e controls. Response t o PHA correlated with t h e absolute T cell values.
.......................................................................................... .......................................................................................... Key words: T and B cells counts, lymphocyte function, PHA response
INTRODUCTION Numerous testing criteria have been established t o aid in management of cancer patients. Unfortunately, none of these procedures is highly definitive, and currently it is not possible t o predict the specific effect of radiation, chemo- or immunotherapy o n resistance of the patient to his neoplasm. Only b y following repeatedly and carefully the From the University of Utah College of Medicine, Medical Center, and Department of Surgery, LDS Hospital, Salt Lake City Utah. Address reprint requests to Charles R. Smart, MD, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT 84143.
0022-4790/79/1103-0217502.00 0 1979 Alan R. Liss, Inc.
218
Hadfield, Marcus, and Smart
changes induced by the treatment in each patient may it be possible t o achieve the desired direction of responsiveness. In vivo testing has shown the lymphocyte to be the cell type responsible for the transfer of tumor resistance from immunized donor t o nonimmune recipient. Furthermore, in vitro tests which assess the ability of sensitized lymphocytes to kill appropriate target cells in tissue culture are now firmly established. It appears that lymphocytes may kill directly [Hellstrom and Hellstrom, 19741 or by the elaboration of cytotoxins [Raben et al, 19761 or they may be involved with an antibody dependent cytolysis [MacLennan and Harding, 19741. The association of lymphocytes with tumor cell destruction and recent publications suggesting a direct relationship between T cell counts and neoplasia [Broeman and Deeley, 1975; Wybran and Fudenberg, 1973; Wybran and Fudenberg, 19711 provide a basis for exploring the hypothesis that T cell values could be used to monitor the immune competence of cancer patients.
MATERIALS AND METHODS Selection of Patient
Patients were selected if they had received no therapy or if they were postsurgery but preadjunctive therapy. The patients had either carcinoma of the breast, pulmonary tree, or gastrointestinal tract. Control patients consisted of patients who were biopsynegative as well as laboratory personnel and outpatients without evidence of tumor.
Blood Collection Twenty milliliters of blood was collected in heparinized vacutainer tubes and processed within three hours of collection. One tube containing anticoagulant ethylenediamine tetraacetic acid (EDTA) was collected for blood counts, and slides were prepared for differential values. Complete blood counts were done on a Coulter Senior blood counter and the differential values were determined by hematology laboratory personnel. Processing of T Cells and B Cells
Lymphocytes were separated from whole blood by a modified method of Boyum [1968]. Specimens were processed on the day drawn and never refrigerated. If drawn in the afternoon the specimens were kept at room temperature overnight and tested in the morning. The heparinized blood was diluted 1 :2 with sterile, pyrogen-free phosphate buffered saline (PBS) in a 50 ml plastic centrifuge tube. Sterile Hypaque Ficoll, 15 ml, was delivered to the bottom of the tube using a Wintrobe tube cannula. The sample was centrifuged for 40 minutes at 400g at room temperature. The resultant band of cells consisted of approximately 90% lymphocytes and 10%monocytes. Lymphocyte recovery was greater than 90% in the preparations tested. The cells were washed three times in PBS and resuspended in RPMI-1640 culture medium. The cells were counted in a hemocytometer and adjusted to desired concentrations.
T Cell Counts Peripheral blood T cell values were determined by the method of Jondall et a1 [ 19721 Two million lymphocytes were mixed with 0.2 ml of a thrice washed 1% sheep red blood cell suspension and incubated at 37OC for 15 minutes. The cells were then centrifuged at
Cancer Therapy on T and B Lymphocytes
219
200g for 5 minutes and refrigerated (2-4°C) for 18 hours. The cells were then resuspended and the number of rosette-forming cells determined by counting 200 or more lymphocytes in a hemocytometer. By convention, any lymphocyte with three or more adherent sheep erythrocytes was considered a T rosette. The number of rosette forming cells was determined with a light microscope at 400 X magnification. All samples throughout the study were read by the same individual without knowledge of specimen identity. T cell values were expressed as a percentage and as the number of T cells/mm3, calculated by the formula: Absolute number of peripheral blood T cells = total leukocyte count X % T cells.
B Cell Counts The population of B cells in the peripheral blood was determined by the method of Hallberg et a1 [1974]. Two million lymphocytes were mixed with 1% sensitized (rabbit anti-ox antibody) ox red blood cells. The cells were refrigerated for 15 minutes and centrifuged at 200g for five minutes. The cells were resuspended and the number of rosette forming cells determined. Again, any lymphocyte with three or more adherent erythrocytes was considered a B rosette. B cell rosettes were determined as described above and were reported as a proportion and as an absolute value.
PHA Stimulation PHA stimulation tests were done to determine whether lymphocytes from patients had the capacity t o respond t o a mitogen and presumably to specific antigens. Washed lymphocytes (1 X 106/ml) were resuspended in WMI-1640 culture medium with 5% pooled human serum and 2 ml of the lymphocyte preparation was added to each of 3 control tubes; 0.4 ml of a 1 : 50 dilution of Difco PHA-P was added to 8 ml of remaining cells, and 2.1 ml of this preparation of cells was pipetted into each of four tubes. The cells were incubated in a 95% air and 5% COz humidified incubator for 48 hours. One microcurie of tritiated thymidine was added to each of the cultured tubes and the cultures were incubated an additional 1 8 hours. After adding cold 5% trichloroacetic acid, the cells were harvested on a millipore sampling manifold. The filter pads were washed twice with cold PBS, then put into 5 ml of aquasol scintillation fluid (NEW) and counted in a Packard liquid scintillation counter. RESULTS Controls Standard hematology tests showed the control population to have a mean WBC count of 7,157, PMN values of 60%, lymphocyte values of 32%,monocyte values of 7%, eosinophile counts of 2%, and basophile counts of less than 1%. No significant differences in the WBC count and relative leukocyte values were observed in the various age groups tested. Patients Patients were categorized according to tumor type. The patients with breast cancer had mean white blood cell counts of 5,731; for pulmonary cancer patients the mean WBC count was 7,014, and for patients with GI tract cancer the mean WBC count was 7,005. The cancer patients had significantly higher PMN values and significantly lower lymphocyte values when compared to the control patients. No significant differences were noted between the monocyte, eosinophile, and basophile leukocytes among the designated groups.
220
Hadfield, Marcus, and Smart
Relative and absolute values for T and B cells and presented in Table I. No significant differences were observed when the relative T cell values were compared among the four groups. However, when the absolute numbers of T cells/mm3 of peripheral blood were compared, a significant decrease in the number of T cells was noted in each of the cancer groups when compared t o the control. These differences were significant at the p = 0.01 level as determined by the Duncan test [Duncan, 19601 for statistical differences. There were no differences when the values were compared among the tumor groups. No significant differences were observed when the relative and absolute numbers of B cells were compared. Although responses to PHA were not statistically significantly different among the four groups, it is evident that the cancer patients yielded less response. The PHA ratio was determined by the following formula: CPM of PHA treated cells/CPM of untreated cells from same patient. Each sample was run in triplicate, and the means were used to determine the PHA ratio. The PHA response data for the group comparisons are shown in Table I. Patients were assigned to treatment groups regardless of their tumor type and comparisons of their leukocyte values were made. The treatment groups were pretherapy, postsurgery preadjunctive therapy, postsurgery postadjunctive therapy, all compared with the nontumor nontreatment control group. Comparison of the WBC counts in the four groups (Table 11) showed the postsurgery postadjunctive therapy group to have a significantly depressed WBC count when compared t o the other groups. All of the treatment groups also had a significantly decreased lymphocyte count when compared t o the nontumor nontreatment control group. No significant differences were noted in the monocyte, eosinophile, or basophile leukocyte counts. Comparison of the relative number of T cells among these groups showed the postsurgery post adjunctive therapy group to be significantly depressed when compared to the other groups. Likewise, when the absolute number of T cells was compared it was found that the postsurgery postadjunctive therapy group had a significantly lower number of T cells/mm3 of peripheral blood (474) when compared to the other groups. In addition it was found that the pretherapy group and the postsurgery preadjunctive therapy group had significantly lower absolute T cell values when compared t o the control group. The postsurgery preadjunctive therapy group had elevated T cell values when compared t o the pretherapy group. These specimens were collected approximately two weeks postsurgery.
TABLE I. Lymphocyte Types and PHA Ratios in Cancer ~~~
Group Control (no tumor or therapy)
Number of patients 76 (26 for B cells)
~
B cells f SE
T cells f SE number" (%)
numbera (%)
1216 + 5 3 (55 f 1)
583 f 50 (30 f 2)
117 f 14 (89 f 143)b
PHA ratio f SE
Breast tumors
65-87
657 f 54 (44 2)
394 f 31 (29 f 2)
84 f 12 (60 f 108)b
Pulmonary tumors
17-21
617 f 9 0 (49 f 4)
399 f 4 3 (36 3)
*
1 O O f 36 (28 f 172)b
GI tract tumors
26- 35
723 f 89 (43 f 3)
404 ? 51 (29 f 3)
84 f 16 (52 1 16)b
aPer mm3 b95% confidence limits.
*
*
75-99
Post surgery, post therapy (all tumors+
70 f 2
aNumber per mm3. bpercent of total. C 9 5 % confidence limits. dThat is, patients with breast, pulmonary, or GI tract tumors in this study.
5672 f 213
6652 f 206 20 f 1
23 f 1
474 2 39 ((41 f 2)
801 f 6 4 (50 f 2)b
75-76
Post surgery, pre therapy (an tumors)d
68fl
703 f 57 (49 f
2222
69 f 2
6880 f 319
34- 37
f SE
1210f53
TI
Pre therapy (an tumor s)d
32 f 1
PMNb f SE Lymphsb f SE
(55 f
WBCa f SE 60 f 1
Number
Control 74-76 7157 ? 184 (no tumor or therapy) (26 for B cells)
Group
TABLE 11. Absolute and Relative White Blood Cell Values in Cancer Groups
331 f 229 (33 f 2)
468 f 47 (32 f 2)b
563 ? 109 (32 f 3)b
584 f 50 (30 f 2)b
B~ f SE
64 (44-83)
121 (89-153)
56 (33-78)
118 (91-145)
PHA ratio f SEC
W
2
V
4
B
c
222
Hadfield, Marcus, and Smart
It was felt that had another sample at a later date but prior t o adjunctive therapy been obtained, these values would have been more elevated and might have reached the control (normal) range. Comparison of the relative number of B cells showed no significant differences; however, when the absolute number of B cells was compared, it was noted that the postsurgery postadjunctive therapy group and the postsurgery preadjunctive therapy group had significantly lower numbers of B cells when compared to the pretherapy and the nontumor nontreatment control group. Comparison of the PHA ratios among the four groups indicated that the pretherapy and the postsurgery postadjunctive therapy groups had significantly lower PHA ratios than the postsurgery preadjunctive therapy group and the nontumor nontreatment control group. These data correlate well with the absolute number of T cells found in the peripheral blood and suggest that 1) therapy reduced T cells in number as well as function, ie, the ability to respond to mitogens and presumably antigens; 2) the tumor has an immunosuppressive effect in that the absolute numbers of T cells are significantly lower than the control values.
DISCUSSION The white blood cell count is often employed to monitor the toxicity of an antitumor regimen. Data presented here suggest that total white cell count is a poor guide t o toxicity because significant differences may not be observed in total count at a time when lymphocytes have been significantly depressed. Our data suggest that the type of tumor, ie carcinoma of the breast, pulmonary tree, or GI tract, may have no significant effect on the absolute white blood cell count. In general, the relative proportion of the leukocytes in this patient sample was altered in that the cancer patients had elevated PMN values and depressed lymphocyte values. Raben et a1 [ 19761 reported data similar to ours, ie, the white blood cell counts in their sample did not vary significantly before radiation therapy in patients with breast or pelvic cancer. The differences in the proportion of PMNs and lymphocytes may be due to a mild inflammatory response at the tumor site. There was no significant difference noted in the relative proportion of T cells among the four groups examined. However, when total numbers of T cells were compared it was obvious that all of the cancer patients had significantly lower T cell values than the control group. Similar data have been reported by Nemoto et a1 [ 19741 in a study on breast cancer patients, although the differences noted in their study were not significant. When the patients were grouped according to the therapy they had received, it became apparent that although the relative proportion of T and B cells did not change significantly with the various treatment modalities, the number of T cells and their ability to respond to PHA were significantly altered. The adjunctive therapy received by the patients consisted of radiation and/or chemotherapy. Campbell et a1 [1976] reported loss of T cells, B cells, and K cells at similar rates during radiation therapy. Likewise, Tarpley et a1 [ 19751 reported destruction of T cells with radiation therapy which was followed by a slow repopulation of the T cells. Patients who were 9 years postradiation therapy still had depressed T cell values and depressed responses to PHA. Raben et a1 119761 found that the levels of total T and B rosettes prior to radiation therapy were similar to those of normal controls. At the completion of the therapy the percent of T rosettes remained unchanged, yet the absolute number of T rosettes decreased significantly when compared t o the normal controls or the patient’s values prior to therapy. Similar data have been reported by Barlow
Cancer Therapy on T and B Lymphocytes
223
et a1 [ 19751 ,Gutierrez et a1 [ 19761 , Cheema and Hersh [ 197 11, and others showing that the chemotherapeutic agents also cause significant depressions in the number and function of T lymphocytes. In contrast, Bloomgren et a1 [1974] have reported an increase in the proportion of T cells as well as that the response t o PHA was not significantly altered and this report agrees with data published by McCredie et a1 [ 19721 . In an extensive study of patients with bronchogenic carcinoma done by Dellon et a1 [1975] it was shown that T cell levels in 12 cancer patients were significantly lower than in 237 controls. Further, the Dellon data show that the T cell levels correlated with the extent of disease and the clinical course of the patient. Like the Dellon group, our data show a decline in the number of T cells in the pretherapy group which suggests that the tumor has an immunosuppressive effect. This is supported by the fact that removal of the tumor mass by surgery results in an increase in the number of T cells. Dellon has shown that T cell levels rise and eventually plateau if adjunctive therapy is not initiated. Our data show an initial rise in T cells following surgery; however, adjunctive therapy was initiated prior to the plateau level. Similarly, our PHA data correlate well with the T cell data suggesting that 1) the tumor causes a decrease in the total numbers of T cells, and 2) the tumor has an immunosuppressive effect on the remaining T cells. Because of the adjunctive therapy intervention we were unable t o use the T cell counts as an indicator of tumor recurrence as Dellon did. Our experience using T cell counts and PHA response as an indicator of therapy toxicity shows that both chemotherapy and radiation or a combination of these modalities are very toxic to T and B cells and critically depress the lymphocyte response t o PHA. Preliminary data suggest that the effects of radiation therapy are slowly overcome whereas the toxicity due to chemotherapy is short-lived, and cell numbers and function return to normal in a short time after discontinuation of the chemotherapy. On a long term basis, ie, after completion of radiotherapy and/or chemotherapy, T cell values and PHA response may be useful in detecting
TABLE 111. Guidelines for Interpretation and Use of
Results (lab report)
Absolute T-cell
PHA
T Cell Counts and Lymphocyte Function Tests Mode of therapy
Therapy that has significant toxic effect on bone marrow
Therapy not affecting bone marrow; may or may not be cytotoxic outside marrow
Immunotherapy, immunostimulation
High High
High Normal
Continue or increase level
Continue or increase level
Value not established
Normal Normal
High Normal
Continue present level
Continue or increase level
Value not established
Normal
LOW
Continue present level
Decrease present level
Recommended
Low Low High
Normal Low Low
Decrease level, intensity or both continue present level
Continue present decrease. level, intensity or both
Recommended Recommended Recommended
Low
High
Decrease level, intensity or both
Continue at present level
Recommended
224
Hadfield, Marcus, and Smart
recurrent disease before it is detectable clinically, as suggested by Dellon’s data as well as the data presented in this paper. It is our opinion that the demonstrated profound immunosuppressive effects of adjunctive radiation and chemotherapy may make it necessary to monitor cellular immune responses for the selection of appropriate adjuvants and regulation of their rate of administration. Quantitative determination of T cells may allow selection of agents which are least immunosuppressive and perhaps immunostimulatory for a given level of tumoricidal effect. Determination of pretreatment cellular immunity and subsequent monitoring of T cell values and PHA response may be a superior method of selecting patients for adjuvant regimes rather than the random allocation of patients t o pre- and postoperative study protocols. On the basis of results reported in the literature and our own experience, we are currently determining the relative validity of the following scheme for interpretation and use of T cell counts and PHA response (Table 111).
ACKNOWLEDGMENTS Excellent technical assistance was provided by John M. Straley.
REFERENCES I . Barlow JE, Harley DL, Fauci AS: Cyclophosphamide suppression o f established cell-mediated immunity. J Clin Invest 56:65-70, 1975. 2. Bloomgren H, Wesserman J , Littbrand B: Blood lymphocytes after radiation therapy of carcinoma of the prostate and urinary bladder. Acta Radiologica 13:357-367, 1974. 3. Boyum A: lsolation of mononuclear cells and granulocytes from human blood. Scand J Lab Clin Invest 21:(suppl97) 77-89, 1968. 4. Broeman J, Deeley TJ: The lymphocyte response and prognosis in cancer of the lung. Br J Radio 48:668-669, 1975. 5. Campbell AC, Wiernik G, Wood J , Hersey P,Waller CA, MacLennan ICM: Characteristics of the lymphopenid induced by radiotherapy. Clin Exp Immunol 23: 200-208, 1976. 6. Cheemd AR, Hersh EM: Patient survival after chemotherapy and its relationship t o in vitro lymphocyte blastogenesis. Cancer 28:851-855, 1971. 7 . Dellon AL, Potvin C, Chretien PB: Thymusdependent lymphocyte levels in bronchogenic cardinoma: Correlations with histology, clinical stage, and clinical course after surgical treatment. Cancer 35:687-694,1975. 8. Duncan DB: Principals and procedures of statistics. Steel RGD, Torrie JH (eds), New York: McGraw-Hill, 1960, pp 107- 109. 9. Gutierrez C, Papamichail M, Pagefaulk W: The effect of cytochalasin B and vinca alkaloids on EA and EAC rosette formation and on the binding of heat aggregated human IgG by human lymphocytes. Clin Exp Immunol23:258--263, 1976 10. Hallberg T, Gurner BW, Coombs KKA: Opsonic adherence o f sensitized ox red blood cells to human lymphocytes as measured by rosette formation. Int Arch Allergy 44:500-514, 1974. 11. Hellstrom I, Hellstrom KE: Cell mediated immune reactions t o tumor antigens with particular emphasis on immunity to human neoplasms. Cancer 34: 1461-1468,1974. 12. Jondal M, Holm G, Wiggell H: Surface markers on human T and B lymphocytes. J Exp Med 136~207-215,1972. 13. MacLennan ICM, Harding B: Non-T cytotoxicity in vitro. In Brent L, Holborow J (eds): “Progress in Immunology I1 ” Amsterdam: North-Holland, 1974, vol 3, pp 347-350. 14. McCredie JA, Inch WR, Sutherland RM: Effect of postoperative radiotherapy on peripheral blood lymphocytes in patients with carcinoma of the breast. Cancer 29:349-356, 1972. 15. Nemoto T , Han T, Minowada J, Angleur V,Chamberlain A, Dao TL: Cell mediated immune status of breast cancer patients: Evaluation b y skin tests, lymphocyte stimulation and counts of rosette forming cells. J Natl Cancer lnst 53:641-645, 1974.
Cancer Therapy on T and B Lymphocytes
225
16. Raben M, Walch N, Galili U,Schlessinger M: The effect of radiation therapy on lymphocyte subpopulations in cancer patients. Cancer 37:1417-1421, 1976. 17. Tarpley JL, Potvin C, Chretien PB: Prolonged depression of cellular immunity in cured laryngopharyngeal cancer patients treated with radiation therapy. Cancer 35:638-644, 1975. 18. Wybran I , Fudenberg HH: Thymus derived rosette forming cells in various human disease states: Cancer, lymphoma, bacterial and viral infections and other diseases. J Clin Invest 52: 1026- 1032, 1973. 19. Wybran J, Fudenberg HH: Rosette formation: A test for cellular immunity. Trans Assoc Am Physicians Phila 84:239-247, 1971.
The Effect of Anticancer Therapy on Peripheral Blood T and B Lymphocyte Counts and Function .......................................................................................... .......................................................................................... T. L. HADFIELD, PhD, S. MARCUS,
PhD, and C.
R. SMART, MD
Patients categorized according t o tumor type were compared to a control nontumor population. Comparison of relative T cell values among the groups showed no significant differences; however, when absolute numbers of T cells/ mm3 were compared, all cancer patients, whether from treated or untreated groups, had significantly depressed T cell values. No significant differences were observed in the relative or absolute numbers of B cells. Comparison of the total lymphocyte response t o PHA showed no significant differences among the various cancer groups; however, response in all cancer groups whether from treated or untreated patients, was depressed by comparison to the control group. Patients categorized according to the type of treatment received showed significant depression in the white blood cell count, lymphocyte count, relative and absolute T cell counts and t h e absolute B cell count in the postsurgery, postadjunctive therapy group. T h e pretherapy group also showed significant depression in the absolute number of T cells/mm3 when compared t o t h e controls. Response t o PHA correlated with t h e absolute T cell values.
.......................................................................................... .......................................................................................... Key words: T and B cells counts, lymphocyte function, PHA response
INTRODUCTION Numerous testing criteria have been established t o aid in management of cancer patients. Unfortunately, none of these procedures is highly definitive, and currently it is not possible t o predict the specific effect of radiation, chemo- or immunotherapy o n resistance of the patient to his neoplasm. Only b y following repeatedly and carefully the From the University of Utah College of Medicine, Medical Center, and Department of Surgery, LDS Hospital, Salt Lake City Utah. Address reprint requests to Charles R. Smart, MD, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT 84143.
0022-4790/79/1103-0217502.00 0 1979 Alan R. Liss, Inc.
218
Hadfield, Marcus, and Smart
changes induced by the treatment in each patient may it be possible t o achieve the desired direction of responsiveness. In vivo testing has shown the lymphocyte to be the cell type responsible for the transfer of tumor resistance from immunized donor t o nonimmune recipient. Furthermore, in vitro tests which assess the ability of sensitized lymphocytes to kill appropriate target cells in tissue culture are now firmly established. It appears that lymphocytes may kill directly [Hellstrom and Hellstrom, 19741 or by the elaboration of cytotoxins [Raben et al, 19761 or they may be involved with an antibody dependent cytolysis [MacLennan and Harding, 19741. The association of lymphocytes with tumor cell destruction and recent publications suggesting a direct relationship between T cell counts and neoplasia [Broeman and Deeley, 1975; Wybran and Fudenberg, 1973; Wybran and Fudenberg, 19711 provide a basis for exploring the hypothesis that T cell values could be used to monitor the immune competence of cancer patients.
MATERIALS AND METHODS Selection of Patient
Patients were selected if they had received no therapy or if they were postsurgery but preadjunctive therapy. The patients had either carcinoma of the breast, pulmonary tree, or gastrointestinal tract. Control patients consisted of patients who were biopsynegative as well as laboratory personnel and outpatients without evidence of tumor.
Blood Collection Twenty milliliters of blood was collected in heparinized vacutainer tubes and processed within three hours of collection. One tube containing anticoagulant ethylenediamine tetraacetic acid (EDTA) was collected for blood counts, and slides were prepared for differential values. Complete blood counts were done on a Coulter Senior blood counter and the differential values were determined by hematology laboratory personnel. Processing of T Cells and B Cells
Lymphocytes were separated from whole blood by a modified method of Boyum [1968]. Specimens were processed on the day drawn and never refrigerated. If drawn in the afternoon the specimens were kept at room temperature overnight and tested in the morning. The heparinized blood was diluted 1 :2 with sterile, pyrogen-free phosphate buffered saline (PBS) in a 50 ml plastic centrifuge tube. Sterile Hypaque Ficoll, 15 ml, was delivered to the bottom of the tube using a Wintrobe tube cannula. The sample was centrifuged for 40 minutes at 400g at room temperature. The resultant band of cells consisted of approximately 90% lymphocytes and 10%monocytes. Lymphocyte recovery was greater than 90% in the preparations tested. The cells were washed three times in PBS and resuspended in RPMI-1640 culture medium. The cells were counted in a hemocytometer and adjusted to desired concentrations.
T Cell Counts Peripheral blood T cell values were determined by the method of Jondall et a1 [ 19721 Two million lymphocytes were mixed with 0.2 ml of a thrice washed 1% sheep red blood cell suspension and incubated at 37OC for 15 minutes. The cells were then centrifuged at
Cancer Therapy on T and B Lymphocytes
219
200g for 5 minutes and refrigerated (2-4°C) for 18 hours. The cells were then resuspended and the number of rosette-forming cells determined by counting 200 or more lymphocytes in a hemocytometer. By convention, any lymphocyte with three or more adherent sheep erythrocytes was considered a T rosette. The number of rosette forming cells was determined with a light microscope at 400 X magnification. All samples throughout the study were read by the same individual without knowledge of specimen identity. T cell values were expressed as a percentage and as the number of T cells/mm3, calculated by the formula: Absolute number of peripheral blood T cells = total leukocyte count X % T cells.
B Cell Counts The population of B cells in the peripheral blood was determined by the method of Hallberg et a1 [1974]. Two million lymphocytes were mixed with 1% sensitized (rabbit anti-ox antibody) ox red blood cells. The cells were refrigerated for 15 minutes and centrifuged at 200g for five minutes. The cells were resuspended and the number of rosette forming cells determined. Again, any lymphocyte with three or more adherent erythrocytes was considered a B rosette. B cell rosettes were determined as described above and were reported as a proportion and as an absolute value.
PHA Stimulation PHA stimulation tests were done to determine whether lymphocytes from patients had the capacity t o respond t o a mitogen and presumably to specific antigens. Washed lymphocytes (1 X 106/ml) were resuspended in WMI-1640 culture medium with 5% pooled human serum and 2 ml of the lymphocyte preparation was added to each of 3 control tubes; 0.4 ml of a 1 : 50 dilution of Difco PHA-P was added to 8 ml of remaining cells, and 2.1 ml of this preparation of cells was pipetted into each of four tubes. The cells were incubated in a 95% air and 5% COz humidified incubator for 48 hours. One microcurie of tritiated thymidine was added to each of the cultured tubes and the cultures were incubated an additional 1 8 hours. After adding cold 5% trichloroacetic acid, the cells were harvested on a millipore sampling manifold. The filter pads were washed twice with cold PBS, then put into 5 ml of aquasol scintillation fluid (NEW) and counted in a Packard liquid scintillation counter. RESULTS Controls Standard hematology tests showed the control population to have a mean WBC count of 7,157, PMN values of 60%, lymphocyte values of 32%,monocyte values of 7%, eosinophile counts of 2%, and basophile counts of less than 1%. No significant differences in the WBC count and relative leukocyte values were observed in the various age groups tested. Patients Patients were categorized according to tumor type. The patients with breast cancer had mean white blood cell counts of 5,731; for pulmonary cancer patients the mean WBC count was 7,014, and for patients with GI tract cancer the mean WBC count was 7,005. The cancer patients had significantly higher PMN values and significantly lower lymphocyte values when compared to the control patients. No significant differences were noted between the monocyte, eosinophile, and basophile leukocytes among the designated groups.
220
Hadfield, Marcus, and Smart
Relative and absolute values for T and B cells and presented in Table I. No significant differences were observed when the relative T cell values were compared among the four groups. However, when the absolute numbers of T cells/mm3 of peripheral blood were compared, a significant decrease in the number of T cells was noted in each of the cancer groups when compared t o the control. These differences were significant at the p = 0.01 level as determined by the Duncan test [Duncan, 19601 for statistical differences. There were no differences when the values were compared among the tumor groups. No significant differences were observed when the relative and absolute numbers of B cells were compared. Although responses to PHA were not statistically significantly different among the four groups, it is evident that the cancer patients yielded less response. The PHA ratio was determined by the following formula: CPM of PHA treated cells/CPM of untreated cells from same patient. Each sample was run in triplicate, and the means were used to determine the PHA ratio. The PHA response data for the group comparisons are shown in Table I. Patients were assigned to treatment groups regardless of their tumor type and comparisons of their leukocyte values were made. The treatment groups were pretherapy, postsurgery preadjunctive therapy, postsurgery postadjunctive therapy, all compared with the nontumor nontreatment control group. Comparison of the WBC counts in the four groups (Table 11) showed the postsurgery postadjunctive therapy group to have a significantly depressed WBC count when compared t o the other groups. All of the treatment groups also had a significantly decreased lymphocyte count when compared t o the nontumor nontreatment control group. No significant differences were noted in the monocyte, eosinophile, or basophile leukocyte counts. Comparison of the relative number of T cells among these groups showed the postsurgery post adjunctive therapy group to be significantly depressed when compared to the other groups. Likewise, when the absolute number of T cells was compared it was found that the postsurgery postadjunctive therapy group had a significantly lower number of T cells/mm3 of peripheral blood (474) when compared to the other groups. In addition it was found that the pretherapy group and the postsurgery preadjunctive therapy group had significantly lower absolute T cell values when compared t o the control group. The postsurgery preadjunctive therapy group had elevated T cell values when compared t o the pretherapy group. These specimens were collected approximately two weeks postsurgery.
TABLE I. Lymphocyte Types and PHA Ratios in Cancer ~~~
Group Control (no tumor or therapy)
Number of patients 76 (26 for B cells)
~
B cells f SE
T cells f SE number" (%)
numbera (%)
1216 + 5 3 (55 f 1)
583 f 50 (30 f 2)
117 f 14 (89 f 143)b
PHA ratio f SE
Breast tumors
65-87
657 f 54 (44 2)
394 f 31 (29 f 2)
84 f 12 (60 f 108)b
Pulmonary tumors
17-21
617 f 9 0 (49 f 4)
399 f 4 3 (36 3)
*
1 O O f 36 (28 f 172)b
GI tract tumors
26- 35
723 f 89 (43 f 3)
404 ? 51 (29 f 3)
84 f 16 (52 1 16)b
aPer mm3 b95% confidence limits.
*
*
75-99
Post surgery, post therapy (all tumors+
70 f 2
aNumber per mm3. bpercent of total. C 9 5 % confidence limits. dThat is, patients with breast, pulmonary, or GI tract tumors in this study.
5672 f 213
6652 f 206 20 f 1
23 f 1
474 2 39 ((41 f 2)
801 f 6 4 (50 f 2)b
75-76
Post surgery, pre therapy (an tumors)d
68fl
703 f 57 (49 f
2222
69 f 2
6880 f 319
34- 37
f SE
1210f53
TI
Pre therapy (an tumor s)d
32 f 1
PMNb f SE Lymphsb f SE
(55 f
WBCa f SE 60 f 1
Number
Control 74-76 7157 ? 184 (no tumor or therapy) (26 for B cells)
Group
TABLE 11. Absolute and Relative White Blood Cell Values in Cancer Groups
331 f 229 (33 f 2)
468 f 47 (32 f 2)b
563 ? 109 (32 f 3)b
584 f 50 (30 f 2)b
B~ f SE
64 (44-83)
121 (89-153)
56 (33-78)
118 (91-145)
PHA ratio f SEC
W
2
V
4
B
c
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Hadfield, Marcus, and Smart
It was felt that had another sample at a later date but prior t o adjunctive therapy been obtained, these values would have been more elevated and might have reached the control (normal) range. Comparison of the relative number of B cells showed no significant differences; however, when the absolute number of B cells was compared, it was noted that the postsurgery postadjunctive therapy group and the postsurgery preadjunctive therapy group had significantly lower numbers of B cells when compared to the pretherapy and the nontumor nontreatment control group. Comparison of the PHA ratios among the four groups indicated that the pretherapy and the postsurgery postadjunctive therapy groups had significantly lower PHA ratios than the postsurgery preadjunctive therapy group and the nontumor nontreatment control group. These data correlate well with the absolute number of T cells found in the peripheral blood and suggest that 1) therapy reduced T cells in number as well as function, ie, the ability to respond to mitogens and presumably antigens; 2) the tumor has an immunosuppressive effect in that the absolute numbers of T cells are significantly lower than the control values.
DISCUSSION The white blood cell count is often employed to monitor the toxicity of an antitumor regimen. Data presented here suggest that total white cell count is a poor guide t o toxicity because significant differences may not be observed in total count at a time when lymphocytes have been significantly depressed. Our data suggest that the type of tumor, ie carcinoma of the breast, pulmonary tree, or GI tract, may have no significant effect on the absolute white blood cell count. In general, the relative proportion of the leukocytes in this patient sample was altered in that the cancer patients had elevated PMN values and depressed lymphocyte values. Raben et a1 [ 19761 reported data similar to ours, ie, the white blood cell counts in their sample did not vary significantly before radiation therapy in patients with breast or pelvic cancer. The differences in the proportion of PMNs and lymphocytes may be due to a mild inflammatory response at the tumor site. There was no significant difference noted in the relative proportion of T cells among the four groups examined. However, when total numbers of T cells were compared it was obvious that all of the cancer patients had significantly lower T cell values than the control group. Similar data have been reported by Nemoto et a1 [ 19741 in a study on breast cancer patients, although the differences noted in their study were not significant. When the patients were grouped according to the therapy they had received, it became apparent that although the relative proportion of T and B cells did not change significantly with the various treatment modalities, the number of T cells and their ability to respond to PHA were significantly altered. The adjunctive therapy received by the patients consisted of radiation and/or chemotherapy. Campbell et a1 [1976] reported loss of T cells, B cells, and K cells at similar rates during radiation therapy. Likewise, Tarpley et a1 [ 19751 reported destruction of T cells with radiation therapy which was followed by a slow repopulation of the T cells. Patients who were 9 years postradiation therapy still had depressed T cell values and depressed responses to PHA. Raben et a1 119761 found that the levels of total T and B rosettes prior to radiation therapy were similar to those of normal controls. At the completion of the therapy the percent of T rosettes remained unchanged, yet the absolute number of T rosettes decreased significantly when compared t o the normal controls or the patient’s values prior to therapy. Similar data have been reported by Barlow
Cancer Therapy on T and B Lymphocytes
223
et a1 [ 19751 ,Gutierrez et a1 [ 19761 , Cheema and Hersh [ 197 11, and others showing that the chemotherapeutic agents also cause significant depressions in the number and function of T lymphocytes. In contrast, Bloomgren et a1 [1974] have reported an increase in the proportion of T cells as well as that the response t o PHA was not significantly altered and this report agrees with data published by McCredie et a1 [ 19721 . In an extensive study of patients with bronchogenic carcinoma done by Dellon et a1 [1975] it was shown that T cell levels in 12 cancer patients were significantly lower than in 237 controls. Further, the Dellon data show that the T cell levels correlated with the extent of disease and the clinical course of the patient. Like the Dellon group, our data show a decline in the number of T cells in the pretherapy group which suggests that the tumor has an immunosuppressive effect. This is supported by the fact that removal of the tumor mass by surgery results in an increase in the number of T cells. Dellon has shown that T cell levels rise and eventually plateau if adjunctive therapy is not initiated. Our data show an initial rise in T cells following surgery; however, adjunctive therapy was initiated prior to the plateau level. Similarly, our PHA data correlate well with the T cell data suggesting that 1) the tumor causes a decrease in the total numbers of T cells, and 2) the tumor has an immunosuppressive effect on the remaining T cells. Because of the adjunctive therapy intervention we were unable t o use the T cell counts as an indicator of tumor recurrence as Dellon did. Our experience using T cell counts and PHA response as an indicator of therapy toxicity shows that both chemotherapy and radiation or a combination of these modalities are very toxic to T and B cells and critically depress the lymphocyte response t o PHA. Preliminary data suggest that the effects of radiation therapy are slowly overcome whereas the toxicity due to chemotherapy is short-lived, and cell numbers and function return to normal in a short time after discontinuation of the chemotherapy. On a long term basis, ie, after completion of radiotherapy and/or chemotherapy, T cell values and PHA response may be useful in detecting
TABLE 111. Guidelines for Interpretation and Use of
Results (lab report)
Absolute T-cell
PHA
T Cell Counts and Lymphocyte Function Tests Mode of therapy
Therapy that has significant toxic effect on bone marrow
Therapy not affecting bone marrow; may or may not be cytotoxic outside marrow
Immunotherapy, immunostimulation
High High
High Normal
Continue or increase level
Continue or increase level
Value not established
Normal Normal
High Normal
Continue present level
Continue or increase level
Value not established
Normal
LOW
Continue present level
Decrease present level
Recommended
Low Low High
Normal Low Low
Decrease level, intensity or both continue present level
Continue present decrease. level, intensity or both
Recommended Recommended Recommended
Low
High
Decrease level, intensity or both
Continue at present level
Recommended
224
Hadfield, Marcus, and Smart
recurrent disease before it is detectable clinically, as suggested by Dellon’s data as well as the data presented in this paper. It is our opinion that the demonstrated profound immunosuppressive effects of adjunctive radiation and chemotherapy may make it necessary to monitor cellular immune responses for the selection of appropriate adjuvants and regulation of their rate of administration. Quantitative determination of T cells may allow selection of agents which are least immunosuppressive and perhaps immunostimulatory for a given level of tumoricidal effect. Determination of pretreatment cellular immunity and subsequent monitoring of T cell values and PHA response may be a superior method of selecting patients for adjuvant regimes rather than the random allocation of patients t o pre- and postoperative study protocols. On the basis of results reported in the literature and our own experience, we are currently determining the relative validity of the following scheme for interpretation and use of T cell counts and PHA response (Table 111).
ACKNOWLEDGMENTS Excellent technical assistance was provided by John M. Straley.
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16. Raben M, Walch N, Galili U,Schlessinger M: The effect of radiation therapy on lymphocyte subpopulations in cancer patients. Cancer 37:1417-1421, 1976. 17. Tarpley JL, Potvin C, Chretien PB: Prolonged depression of cellular immunity in cured laryngopharyngeal cancer patients treated with radiation therapy. Cancer 35:638-644, 1975. 18. Wybran I , Fudenberg HH: Thymus derived rosette forming cells in various human disease states: Cancer, lymphoma, bacterial and viral infections and other diseases. J Clin Invest 52: 1026- 1032, 1973. 19. Wybran J, Fudenberg HH: Rosette formation: A test for cellular immunity. Trans Assoc Am Physicians Phila 84:239-247, 1971.
