Int. J. Radiolion
Oncology
Bid
Phy.5
1977, Vol. 2, pp. 289-295.
Pergamon
Press.
Printed m the U.S A
??Original Contribution
BLOOD LYMPHOCYTES IN BREAST CANCER PATIENTS FOLLOWING RADIOTHERAPY AND SURGERY EDWARD BARAL, M.D. Radiumhemmet,
Karolinska
and HENRIC BLOMGREN, M.D. Hospital,
S-104 01 Stockholm,
Sweden
and BJ~RN PETRINI, M.D. The Central
Microbiological
and JERZY WASSERMAN,
Laboratory, helm,
Stockholm
County
Council,
M.D. S-101 22 Stock-
Sweden
Peripheral blood lymphocytes from 110 subjects with primary carcinoma of the breast were studied before and at various times after local radiotherapy and/or surgery. The results demonstrated that the recovery of the relative PPD (purified protein derivative) responses of the lymphocytes after radiotherapy progresses more rapidly than the repopulation of the total lymphocyte population. Radiotherapy,
Breast cancer, Lymphopenia,
INTRODUCTION
radiotherapy for cancer has External been shown to induce a state of peripheral lymphopenia~*.~.~.ll.13.14.1erI.zs.lh We have
Lymphocyte
reactivity, Lymphocyte
stimulation.
In this investigation we have examined the blood lymphoid population in women with primary carcinoma of the breast before and at various intervals after local radiotherapy and/or surgery. It is shown that the recovery of the relative PPD-response of the lymphocytes after radiotherapy progresses more rapidly than the repopulation of the total lymphocyte population.
that lymphoid cell preparaobserved tions obtained from such patients shortly after completion of radiotherapy exhibit principally normal, relative blastogenic response to phytomitogens in vitro, whereas their response to PP D-tuberculin is highly The mechanism for the dedecreased.2.3.y velopment of the longlasting lymphopenia and the reason for the reduction of the PPDresponse of the lymphocytes presently is unknown. Nor is it known whether repopulation of the lymphocyte compartment and the reappearance of PPD-reactivity of the lymphocytes run in parallel or whether there is a dissociation of these two phenomena.
Patients A total number of 110 women with primary carcinoma of the breast was studied. All the patients were included in a clinical trial aimed at establishing the role of pre- and postoperative radiotherapy for prognosis of this disease. Diagnosis was assessed by fine needle aspiration biopsy.’ Operable patients,
Acknowledgements-The authors wish to thank Professor J. Einhorn for his valuable criticism of the manuscript, Mrs. Brita-Maria Berg and Mrs. Ingrid Falk for their excellent technical assistance
and Mr. Stephen Ogenstad for statistical evaluation of the results. The investigation was supported by grants from the King Gustav V Jubilee Fund.
METHODS
AND
MATERIALS
290
Radiation
Oncology
0 Biology
??Physics
less than 70 years of age, free of distant randomized into three metastases were treatment groups: (A) preoperative radiotherapy followed by radical mastectomy, (B) radical mastectomy followed by local radiotherapy and (C) radical mastectomy only. A detailed account of the patients included in this trial, the clinical status of the patients and the randomization procedure have been described before.‘.24 Radiotherapy
Preoperative radiotherapy was given with a “Co source. Treatment was directed to the breast, both parasternal regions, axilla and the supraclavicular fossa. A total target dose of 4500rad was delivered during approximately 6 weeks. Postoperative radiotherapy was given with high energy electrons to the chest wall and the lymph node stations were treated as described above. A total target dose of 4500 rad was delivered during approximately 6 weeks. Details of these techniques have been given before.y.*4 Surgery
All the patients tomy as described Blood
underwent before.9,2”
radical
mastec-
sampling
Blood was collected before and at various times after treatment to establish the number of lymphocytes/p1 and their reactivity to mitogenic stimuli in vitro. The samples were obtained at the following time periods: Treatment group A. Sample number I, was obtained before radiotherapy; sample II, within I month after completion of radiotherapy; sample III, 1 month after surgery; sample IV, 4 months after surgery; sample V, 10 months after surgery; and sample VI, 21-23 months after surgery. Treatment group B. Sample I was obtained before surgery; sample II, 1 month after surgery; and sample III, 1 month after completion of radiotherapy; sample IV, 4 months after radiotherapy; sample V, 10 months after radiotherapy; sample VI, 21-23 months after radiotherapy. Treatment group C. Sample number I was obtained before surgery. Samples II, III, IV,
March-April
1977, Volume
2. No.
3 and No. 4
V and VI were obtained 1 month, 4 months, 7 months, 13 and 24-26 months after surgery, respectively. The patients who developed distant metastases and/or local recurrences during the observation time have been excluded in this report. Separation
of lymphocytes
Venous blood was defibrinated by agitation in beakers containing glass pearls. Lymphoid cells were obtained after gelatin sedimentation of the erythrocytes according to the method described by Co&on and Chalmers.4.5 Lymphocyte
stimulation
tests
These procedures have been described in detail before.’ In short, 2 x 10” lymphoid cells were cultured in glass tubes containing 1.0 ml Eagle’s minimal essential medium supplemented with Earle’s salts (MEM) with 10% heat inactivated human serum, streptomycin and penicillin. To some of the cultures was added phytohaemagglutinin (PHA, Bacto- ’ Phytohaemagglutinin-M, Difco lab. Detroit Mich., USA) at a final concentration of 0.6 mglml and to other PPD-tuberculin (PPD, RT 22, Statens Seruminstitut, Copenhagen, Denmark) at a concentration of 1 pg/ml. Control cultures, which were set up in parallel received no stimulant. After 4 days of incubation at 37°C in a humidified 5% CO,-air atmosphere to each tube was added 0.4 PCi of 14C-thymidine (Radiochemical Center, England, Specific activity Amersham, 54 mCi/ml). Twenty-four hours later the culcompleted and incorporated tures were radioactivity was determined as described before.!’ Radioactivity of the control cultures, expressed as counts per minute (CPM), was subtracted from the values obtained in the corresponding test cultures. Mean values were calculated from triplicate cultures. Statistical
evaluation
of the results
As described above populations of individual at six different occasions of approximately 2 years. reasons we did not obtain
the lymphoid cell patients were tested during a time period For various trivial test results from all
Blood lymphocytes
in breast cancer 0 E.
BARAL
291
et al.
patients at all occasions. Therefore, the mean values and SE. (standard errors), calculated on a geometric basis, which are presented in each diagram in Figs. 1-3, are not based on the same numbers of patients. When evaluating whether there is a statistically significant difference between the values obtained at two test occasions, we only included pairs of observations, i.e. patients who were tested at both times. The difference between the arithmetic mean values were considered significant when the P-values were 50.05. The arithmetic mean values are presented in Tables l-3.
P
Yl
c-1
PI
T
T
*
i
______~____ P
(34
I
m
1
P
Test
II
number
Fig. 2. Lymphocyte numbers and their relative responses to PPD and PHA in vitro in patients receiving postoperative radiotherapy (treatment Group B). The patients were tested at various intervals during approximately 2 years. For details concerning the test periods, see “Methods and Material”. (CPM = counts per minute.)
RESULTS
_~--__---~ P
Test
m
number
Fig. 1. Number of lymphocytes/PI of blood and their relative responses to PPD and PHA in vitro in patients receiving preoperative radiotherapy (treatment Group A). The patients were tested at various intervals during approximately 2 years. For details concerning the test periods, see “Methods and Materials”. Mean values kS.E. are presented. Figures within parentheses indicate the number of patients. (CPM = counts per minute.)
Peripheral lymphocyte population in preoperatively irradiated patients (Group A) The mean lymphocyte counts of all the patients in this group and the relative responses to PPD and PHA in vitro are presented in Fig. 1 and statistical evaluation of the results is shown in Table 1. The number of lymphocytes and their responsiveness to PPD, but not to PHA, were significantly reduced after completion of radiotherapy (test number II). Thereafter the PPD-reactivity increased sharply and reached the original level 5 months after irradiation (test number IV). In contrast, the
292
Radiation
Oncoiogy
0 Biology 0 Physic?
ts
i Yl
% z
E
$
(2’1P P D
I
~26~ T
20+
T
(24:
I
15p f _
1
1
III
m
Ip
I
\2?
(291
i
I L
P
YI
101 i
% 1
8OL (26) PHA 1 '7' 60~~3$_~l $
IF?
(241 f
I
; dOl_ Jr III
Ill
E
Test
P
TLI
number
Fig. 3. Lymphocyte numbers and their relative responses to PPD and PHA in vitro in patients treated with surgery alone (treatment Group C). The patients were tested at intervals during approximately 2 years. For details concerning the test periods, see “Methods and Materials”. (CPM = counts per minute.)
repopulation of lymphocytes proceeded more slowly and the lymphocyte counts were not normalized at the end of the observation period of 2 years. The relative PHA-reactivity of the cells increased after surgery. This enhancement of the response was found to be statistically significant.
March-April
1977. Volume
2. No. 3 and No. 4
Peripheral lymphocyte population in postoperutively irradiated patients (Group B) The results which are shown in Fig. 2 and Table 2 are analogous to those of preoperatively irradiated patients. Thus, there was a statistically significant decrease of both the cell numbers and their PPD-reactivity within 1 month after completion of radiotherapy (test number III). On the other hand their PHA reactivity showed a tendency to a slight, but not significant increase following surgery. Thereafter the PPD reactivity increased more rapidly than the number of lymphocytes. The PHA-responsiveness of the cells started to increase approximately 1 year after treatment and at 2 years (test number VI) the responsiveness was significantly higher than that recorded before treatment (test number I). Peripherul lymphocyte population in patients treated with surgery only (Group C) Figure 3 and Table 3 show that the number of lymphocytes did not change significantly after radical mastectomy. On the other hand, both the PPD- and the PHA-responses of the cells increased after surgery, although the differences were non-significant. However, the relative stimulations obtained before treatment (test number I) were significantly lower than those recorded at the last test. DISCUSSION The results of this investigation confirm and extend our previous observations ob-
Table 1. Statistical evaluation of the lymphocyte counts, PPD and PHA responses at different test periods in patients irradiated preoperatively (Group A). The arithmetic mean values? and the numbers of patientsS are given Test
values
compared5
Lymphocyte
I and II
2020 > 91 It (36)$ p < 0.001 895 < 1540 (28) p < 0.001 2078> 1511 (26)
II and VI I and VI
p < 0.001
counts
PPD responses (CPM)
PHA responses (CPM)
19.634 > 9553 (40) 46.363 < 53.243 p < 0.001 NS 7.639< 18.140 (28) 51.431 ~66.372 p < 0.001 p < 0.05 18.137 p
1.762t NS
I and VI
5Test approximately
counts
I:
PHA
(CPM)
(26) 53.787 < 55.415 NS
(25)
1.788 < 1.896 (22) 20.612 < 29.577 (25) 52.970
Oncology
Bid
Phy.5
1977, Vol. 2, pp. 289-295.
Pergamon
Press.
Printed m the U.S A
??Original Contribution
BLOOD LYMPHOCYTES IN BREAST CANCER PATIENTS FOLLOWING RADIOTHERAPY AND SURGERY EDWARD BARAL, M.D. Radiumhemmet,
Karolinska
and HENRIC BLOMGREN, M.D. Hospital,
S-104 01 Stockholm,
Sweden
and BJ~RN PETRINI, M.D. The Central
Microbiological
and JERZY WASSERMAN,
Laboratory, helm,
Stockholm
County
Council,
M.D. S-101 22 Stock-
Sweden
Peripheral blood lymphocytes from 110 subjects with primary carcinoma of the breast were studied before and at various times after local radiotherapy and/or surgery. The results demonstrated that the recovery of the relative PPD (purified protein derivative) responses of the lymphocytes after radiotherapy progresses more rapidly than the repopulation of the total lymphocyte population. Radiotherapy,
Breast cancer, Lymphopenia,
INTRODUCTION
radiotherapy for cancer has External been shown to induce a state of peripheral lymphopenia~*.~.~.ll.13.14.1erI.zs.lh We have
Lymphocyte
reactivity, Lymphocyte
stimulation.
In this investigation we have examined the blood lymphoid population in women with primary carcinoma of the breast before and at various intervals after local radiotherapy and/or surgery. It is shown that the recovery of the relative PPD-response of the lymphocytes after radiotherapy progresses more rapidly than the repopulation of the total lymphocyte population.
that lymphoid cell preparaobserved tions obtained from such patients shortly after completion of radiotherapy exhibit principally normal, relative blastogenic response to phytomitogens in vitro, whereas their response to PP D-tuberculin is highly The mechanism for the dedecreased.2.3.y velopment of the longlasting lymphopenia and the reason for the reduction of the PPDresponse of the lymphocytes presently is unknown. Nor is it known whether repopulation of the lymphocyte compartment and the reappearance of PPD-reactivity of the lymphocytes run in parallel or whether there is a dissociation of these two phenomena.
Patients A total number of 110 women with primary carcinoma of the breast was studied. All the patients were included in a clinical trial aimed at establishing the role of pre- and postoperative radiotherapy for prognosis of this disease. Diagnosis was assessed by fine needle aspiration biopsy.’ Operable patients,
Acknowledgements-The authors wish to thank Professor J. Einhorn for his valuable criticism of the manuscript, Mrs. Brita-Maria Berg and Mrs. Ingrid Falk for their excellent technical assistance
and Mr. Stephen Ogenstad for statistical evaluation of the results. The investigation was supported by grants from the King Gustav V Jubilee Fund.
METHODS
AND
MATERIALS
290
Radiation
Oncology
0 Biology
??Physics
less than 70 years of age, free of distant randomized into three metastases were treatment groups: (A) preoperative radiotherapy followed by radical mastectomy, (B) radical mastectomy followed by local radiotherapy and (C) radical mastectomy only. A detailed account of the patients included in this trial, the clinical status of the patients and the randomization procedure have been described before.‘.24 Radiotherapy
Preoperative radiotherapy was given with a “Co source. Treatment was directed to the breast, both parasternal regions, axilla and the supraclavicular fossa. A total target dose of 4500rad was delivered during approximately 6 weeks. Postoperative radiotherapy was given with high energy electrons to the chest wall and the lymph node stations were treated as described above. A total target dose of 4500 rad was delivered during approximately 6 weeks. Details of these techniques have been given before.y.*4 Surgery
All the patients tomy as described Blood
underwent before.9,2”
radical
mastec-
sampling
Blood was collected before and at various times after treatment to establish the number of lymphocytes/p1 and their reactivity to mitogenic stimuli in vitro. The samples were obtained at the following time periods: Treatment group A. Sample number I, was obtained before radiotherapy; sample II, within I month after completion of radiotherapy; sample III, 1 month after surgery; sample IV, 4 months after surgery; sample V, 10 months after surgery; and sample VI, 21-23 months after surgery. Treatment group B. Sample I was obtained before surgery; sample II, 1 month after surgery; and sample III, 1 month after completion of radiotherapy; sample IV, 4 months after radiotherapy; sample V, 10 months after radiotherapy; sample VI, 21-23 months after radiotherapy. Treatment group C. Sample number I was obtained before surgery. Samples II, III, IV,
March-April
1977, Volume
2. No.
3 and No. 4
V and VI were obtained 1 month, 4 months, 7 months, 13 and 24-26 months after surgery, respectively. The patients who developed distant metastases and/or local recurrences during the observation time have been excluded in this report. Separation
of lymphocytes
Venous blood was defibrinated by agitation in beakers containing glass pearls. Lymphoid cells were obtained after gelatin sedimentation of the erythrocytes according to the method described by Co&on and Chalmers.4.5 Lymphocyte
stimulation
tests
These procedures have been described in detail before.’ In short, 2 x 10” lymphoid cells were cultured in glass tubes containing 1.0 ml Eagle’s minimal essential medium supplemented with Earle’s salts (MEM) with 10% heat inactivated human serum, streptomycin and penicillin. To some of the cultures was added phytohaemagglutinin (PHA, Bacto- ’ Phytohaemagglutinin-M, Difco lab. Detroit Mich., USA) at a final concentration of 0.6 mglml and to other PPD-tuberculin (PPD, RT 22, Statens Seruminstitut, Copenhagen, Denmark) at a concentration of 1 pg/ml. Control cultures, which were set up in parallel received no stimulant. After 4 days of incubation at 37°C in a humidified 5% CO,-air atmosphere to each tube was added 0.4 PCi of 14C-thymidine (Radiochemical Center, England, Specific activity Amersham, 54 mCi/ml). Twenty-four hours later the culcompleted and incorporated tures were radioactivity was determined as described before.!’ Radioactivity of the control cultures, expressed as counts per minute (CPM), was subtracted from the values obtained in the corresponding test cultures. Mean values were calculated from triplicate cultures. Statistical
evaluation
of the results
As described above populations of individual at six different occasions of approximately 2 years. reasons we did not obtain
the lymphoid cell patients were tested during a time period For various trivial test results from all
Blood lymphocytes
in breast cancer 0 E.
BARAL
291
et al.
patients at all occasions. Therefore, the mean values and SE. (standard errors), calculated on a geometric basis, which are presented in each diagram in Figs. 1-3, are not based on the same numbers of patients. When evaluating whether there is a statistically significant difference between the values obtained at two test occasions, we only included pairs of observations, i.e. patients who were tested at both times. The difference between the arithmetic mean values were considered significant when the P-values were 50.05. The arithmetic mean values are presented in Tables l-3.
P
Yl
c-1
PI
T
T
*
i
______~____ P
(34
I
m
1
P
Test
II
number
Fig. 2. Lymphocyte numbers and their relative responses to PPD and PHA in vitro in patients receiving postoperative radiotherapy (treatment Group B). The patients were tested at various intervals during approximately 2 years. For details concerning the test periods, see “Methods and Material”. (CPM = counts per minute.)
RESULTS
_~--__---~ P
Test
m
number
Fig. 1. Number of lymphocytes/PI of blood and their relative responses to PPD and PHA in vitro in patients receiving preoperative radiotherapy (treatment Group A). The patients were tested at various intervals during approximately 2 years. For details concerning the test periods, see “Methods and Materials”. Mean values kS.E. are presented. Figures within parentheses indicate the number of patients. (CPM = counts per minute.)
Peripheral lymphocyte population in preoperatively irradiated patients (Group A) The mean lymphocyte counts of all the patients in this group and the relative responses to PPD and PHA in vitro are presented in Fig. 1 and statistical evaluation of the results is shown in Table 1. The number of lymphocytes and their responsiveness to PPD, but not to PHA, were significantly reduced after completion of radiotherapy (test number II). Thereafter the PPD-reactivity increased sharply and reached the original level 5 months after irradiation (test number IV). In contrast, the
292
Radiation
Oncoiogy
0 Biology 0 Physic?
ts
i Yl
% z
E
$
(2’1P P D
I
~26~ T
20+
T
(24:
I
15p f _
1
1
III
m
Ip
I
\2?
(291
i
I L
P
YI
101 i
% 1
8OL (26) PHA 1 '7' 60~~3$_~l $
IF?
(241 f
I
; dOl_ Jr III
Ill
E
Test
P
TLI
number
Fig. 3. Lymphocyte numbers and their relative responses to PPD and PHA in vitro in patients treated with surgery alone (treatment Group C). The patients were tested at intervals during approximately 2 years. For details concerning the test periods, see “Methods and Materials”. (CPM = counts per minute.)
repopulation of lymphocytes proceeded more slowly and the lymphocyte counts were not normalized at the end of the observation period of 2 years. The relative PHA-reactivity of the cells increased after surgery. This enhancement of the response was found to be statistically significant.
March-April
1977. Volume
2. No. 3 and No. 4
Peripheral lymphocyte population in postoperutively irradiated patients (Group B) The results which are shown in Fig. 2 and Table 2 are analogous to those of preoperatively irradiated patients. Thus, there was a statistically significant decrease of both the cell numbers and their PPD-reactivity within 1 month after completion of radiotherapy (test number III). On the other hand their PHA reactivity showed a tendency to a slight, but not significant increase following surgery. Thereafter the PPD reactivity increased more rapidly than the number of lymphocytes. The PHA-responsiveness of the cells started to increase approximately 1 year after treatment and at 2 years (test number VI) the responsiveness was significantly higher than that recorded before treatment (test number I). Peripherul lymphocyte population in patients treated with surgery only (Group C) Figure 3 and Table 3 show that the number of lymphocytes did not change significantly after radical mastectomy. On the other hand, both the PPD- and the PHA-responses of the cells increased after surgery, although the differences were non-significant. However, the relative stimulations obtained before treatment (test number I) were significantly lower than those recorded at the last test. DISCUSSION The results of this investigation confirm and extend our previous observations ob-
Table 1. Statistical evaluation of the lymphocyte counts, PPD and PHA responses at different test periods in patients irradiated preoperatively (Group A). The arithmetic mean values? and the numbers of patientsS are given Test
values
compared5
Lymphocyte
I and II
2020 > 91 It (36)$ p < 0.001 895 < 1540 (28) p < 0.001 2078> 1511 (26)
II and VI I and VI
p < 0.001
counts
PPD responses (CPM)
PHA responses (CPM)
19.634 > 9553 (40) 46.363 < 53.243 p < 0.001 NS 7.639< 18.140 (28) 51.431 ~66.372 p < 0.001 p < 0.05 18.137 p
1.762t NS
I and VI
5Test approximately
counts
I:
PHA
(CPM)
(26) 53.787 < 55.415 NS
(25)
1.788 < 1.896 (22) 20.612 < 29.577 (25) 52.970
