Cancer ImmunolImmunother(1991) 33:121 - 127 034070049100040P
~
ancer mmunology mmunotherapy
© Springer-Verlag 1991
Soluble interleukin-2 receptor and soluble CD8 antigen levels in serum from patients with non-resectable lung cancer Jette Vibe-Petersen 1, Niels Tvede 1, Marcus Diamant 1, Anne Arnt Kjerulff I, Hans Rahbek S0rensen 2, and Vagn Andersen 1 1Laboratoryof MedicalImmunology,Departmentof MedicineTTA, Rigshospitalet,Universityof Copenhagen,Denmark 2 Departmentof Thoracic SurgeryRT, Rigshospitalet,Universityof Copenhagen,Denmark Received 6 December 1990/Accepted 17 December 1990
Summary. In a preliminary longitudinal study two women with histologically verified adenocarcinoma of the lung, without simultaneous infectious or inflammatory conditions, were seen every 2 weeks until death. In one of the patients serum soluble interleukin-2 receptor (sIL-2R) levels rose progressively while the levels for the other patient increased during the second half of the observation period. Serum soluble CD8 antigen (sCD8 Ag) showed a pattern dissimilar to the one for sIL-2R. In a retrospective cross-sectional study circulating levels of sIL-2R and sCD8 Ag were measured before explorative thoracotomy in a total of 65 patients with histologically proven non-resectable carcinoma of the lung. The sIL-2R levels were significantly increased independently of histological subclassification while sCD8 Ag was increased only in patients with small-cell lung cancer. There was no correlation between pre-operative values and length of survival. Key words" Lung cancer - sIL-2R - Cytokines - CD8 antigen
Introduction Primary lung cancer is the most frequent malignant condition in men and occurs with increasing frequency in women. Four main histological types of bronchial carcinoma are recognized: squamous cell carcinoma (WHO l c l ) 44%, small-cell carcinoma (WHO lc2) 18%, adenocarcinoma (WHO lc3) 28% and large-cell carcinoma (WHO lc4) 10% [8]. The majority of patients with lung cancer have non-resectable tumours at the time of diagnosis and their prognosis is dismal. Except in patients with small-cell lung cancer
Offprint requests to: J. Vibe-Petersen, StenoMemorialHospital, 2 Niels Steensens Vej, DK-2820Gentofte,Denmark
systemic therapy with cytostatic agents remains experimental [22]. Therefore, new systemic therapies are badly needed. Human interleukin-2 (IL-2) is a 15-kDa glycosylated protein produced by a subset of activated T lymphocytes of helper cell phenotype. In recent clinical trials [9] adoptive transfer of IL-2 and cells with lymphokine-activated killer activity has evoked some responses in patients with melanoma, renal cell carcinoma, colon carcinoma and nonHodgkin's lymphoma. The therapeutic efficacy of this therapy in lung carcinoma has been studied in only a few patients. Those patients with advanced lung cancer responded poorly [27]. IL-2-expanded tumour-infiltrating lymphocytes, administered without systemic IL-2 to patients with lung carcinoma, gave no objective clinical responses [9]. To exert its biological effects, IL-2 must interact with a specific membrane IL-2 receptor (IL-2R) [21], which is expressed on activated normal peripheral blood mononuclear cells (PBMC: T cells, B cells, NK cells and monocytes). Using two monoclonal antibodies, anti-Tac and 7G7/B6 [17], directed against different epitopes on the Tac peptide, Rubin et al. developed an enzyme-linked immunoabsorbent assay to measure soluble IL-2R (sIL-2R; soluble Tac antigen) [16]. Activated PBMC as well as certain T and B cell lines were found to release soluble IL-2R into the culture medium [4, 7, 16]. The soluble form of the receptor (40-45 kDa) is smaller than its cellular counterpart but retains the ability to bind IL-2 [16]. Soluble Tac peptide is found in the serum of normal individuals and at elevated levels in the serum of patients with a variety of lymphoproliferative and haematological malignancies [11]. The CD8 antigen has long been used as a surface marker for the identification of cytotoxic and suppressor cells. Recently CD8-positive cells have been shown similarly to release a soluble form of the CD8 antigen (sCD8 Ag) [23], a 52-kDa homodimer, in response to actiration. The concentration of sCD8 Ag in serum has been shown to be elevated in various pediatric lymphoid malignancies [13, 14].
122 Table 1. Sequential registration of clinical parameters in two patients with pulmonary adenocarcinomaa Parameter
Experimental time (weeks) 0
2
4
65.0 -
63.5 -
62.5 -
NC Left humerus
NC
54.5
53.5
6
8
10
12
14
16
18
47.5
47.0
Patient 1 Weight (kg) Episodes of fever Chest X-ray Metastases
refused further weight control --
NC Right humerus
6 cutaneous
+
Size ?
Dead week 13
Size$'l" 2 new
Patient 2 Weight (kg) Episodes of fever Chest X-ray
59.0
Metastases
X-ray: os-~ coccygis
53.5
49.0
Dead week 19b
m
PD minor
PD PD minor single-shot irradiation
PD
NC
PD fast
PD Hepatic
NC, no change; PD, progressive disease (~>25%) b Autopsy showed pulmonary carcinoma and metastases in the lymph nodes, liver and lumbar spine
The present study was designed (a) to evaluate a sequential examination of both immunological and clinical parameters in two patients with lung cancer with one ageand sex-matched control person with blood samples drawn every 2 weeks; (b) on the basis of these data to pefform a cross-sectional examination of sIL-2R and CD8 Ag in serum samples collected before explorative thoracotomy from 65 patients with non-resectable carcinoma of the lung; (c) to estimate a possible prognostic correlation between pre-operative values of sIL-2R/sCD8 Ag and survival.
Materials and methods Study subjects. Two women with bioptically verified adenocarcinoma of the lung (WHO lc3) and one age- and sex-matched control person (respective ages 57, 60 and 56 years) were simultaneously included in this preliminaryprospective longitudinal study. The diagnosis was established by an explorative thoracotomy approximately 14 days before entry and the patients received no treatment with glucocorticoids or cytotoxic agents. They were seen every fortnight and the following clinical parameters were registered: weight, clinical examinaton, medicine consumption and episodes of fever. Every time the patient visited the out-patient clinic, a chest X-ray was taken as well as a blood sample with the following distribution: 10 ml for serum (samples stored at -80 ° C), 5 ml for plasma (Iithium heparin, stored at -80 ° C) and 5 ml for values of complete blood counts (haemoglobin, white blood cells, differential counts and platelets). The sequential evaluation of immune parameters included the following: circulating serum levels of IL-2, interferon 7 (IFN7), interleukin-6 (IL-6), tumour necrosis factor-o~ (TNFc~), a soluble or shed form of the IL-2 receptor (sIL-2R) and a soluble form of the CD8 antigen (sCD8 Ag). Informed consent from the patients, according to the Helsinki declaration, was obtained in each case. In addition, a retrospective cross-sectional examination was performed on serum samples from 65 patients with histologically proven non-resectable carcinoma of the lung. Entry criteria included no previous or concurrent malignancies as well as no previous chemotherapy/radiotherapy. The serum samples were collected before explorative thoracotomy, in the period from December 1982 until February 1988 (L. Ols-
son and H. Rahbek S0rensen), and stored at -80°C before analysis of sIL-2R and sCD8 Ag. A group of 11 age-matched healthy donors served as controls.
IL-2, IFN7, sIL-2R, sCD8 Ag and TNFo~ enzyme-linked immunosorbent assay (ELISA). IL-2 levels were determined in previously unthawed heparin-stabilised plasma while IFN7, sIL-2R, sCD8 Ag and TNFc~ levels were measured in unthawed serum samples. Fibrin was removed by incubation of plasma with CaCI2 for 2 h at 37 ° C followed by centrifugation. Plasma was diluted 1 : 2 by phosphatebuffered saline. In order to minimize proteolysis of human IFN7 in test samples, EDTA was added (10 txI EDTA/150 gl sample) for 30 min at room temperature. All parameters were measured using commercially available solidphase sandwich ELISA. The IL-2 ELISA (Intertest-2, Genzyme Corp., Boston, MA 02111, USA) was dependent on a coating antibody recognizing orte epitope on the human IL-2 molecule, which was absorbed onto polysterene microtiter plates. IL-2 present was bound, whereas umeactive components were removed by washing. An enzyme-conjugated antibody recognizing a different epitope on the human IL-2 was added and bound to solid-phase-bound IL-2. Unbound enzyme-conjugated anti-IL-2 was removed by subsequent washing, and a substrate solution was added to form a coloured product proportional to the amount of IL-2 present in the sample. The absorbance was read against a blank using a microtiter ELISA reader. Sample concentrations were determined in duplicate from a standard curve. The other assays were in principle similar to the IL-2 ELISA just described. IFN7 ELISA (Holland Biotechnology BV, Leiden) was dependent on a monoclonal antibody to human IFN7 and to a detector complex consisting of a biotinylated secondary monoclonal antibody to IFN7 and an antibiotin-aikaline-phosphatase conjugate, sIL-2R ELISA (T cell Sciences Inc., Cambridge, MA 02139, USA) was based on the binding of sIL-2R to a solid-phase-bound antibody recognizing the Tac epitope on the human IL-2R [16] and detection of the bound antigen by a horseradish-peroxidase-conjugatedantibody recognizing a different epitope on the human IL-2R (7G7/B6) [17]. sCD8 Ag ELISA (T Cell Sciences Inc., Cambridge, MA 02 139, USA) was dependent on the availability of antibody anti-CD8 mAb C9 and a second horseradish-peroxidase-conjugated anti-CD8 mAb B 12 recognizing a different epitope on the human sCD8 [23]. The TNF« Test Kit (T Cell Sciences Inc., Cambridge, MA 02 139, USA) was based on a murine mAb to human TNFc~ and a secondary horseradish-peroxidase-conjugatedmurine mAb to human TNFo«
123 • • • • Patient
No
1
---
No
2
Patient
Control
11~m
•
. . . "
9-
.
"0"
•
•
~
~
~
Upper
normal
limit
Lower
normal
limit
Upper
normal
limit
Upper
normal
limit
/
7-1
4-
3~m oo
• .i..
2-
Ex _1
10
oO
.
0 v-
•
.......
0...
• .....
..
.
"0
.....
Q•2~. ~..1
700•
°,
.
, •"
•
"0"
•-
600-
e.
x qJ m m 0.
500400-
300-
• 7._..,-..:.i....:I.:
•_.t:'«'..
--
"8.
--..
_,
Lower
normal
limit
"I"
0'
'
4'
'
8
'
1=2
'
1'6
IL-6 bioassay. IL-6 activity was determined using the IL-6-dependent mouse hybridoma cell line B 13.29 clone B9. The B9 hybridoma cells were maintained in culture medium supplemented with 5% heat-inactivated fetal calf serum (Gibco) and 20 U/tal recombinant human (th) IL-6. B9 cells used in the assay were washed three times in IL-6-free medium before culturing. Samples of 5 x 103 B9 cells were cultured together with serial dilutions of the test sample in a final volurne of 200 gl in flat-bottomed microtiter plates. After 68 h 0.5 gCi/well [3H]thymidine was added. After 5 h the cells were harvested on glassfibre filters and the [3H]thymidine incorporation was measured. All assays were performed in triplicate, and 1 U/tal IL-6 activity was defined as the concentration resulting in half-maximal [3H]thymidineincorporation. rIL-6 was used as an internal standard in all assays. Statistics. Unpaired observations were tested by the Mann-Whitney test;
paired observations by the Wilcoxon/Pratt test. The level of significance was P
~
ancer mmunology mmunotherapy
© Springer-Verlag 1991
Soluble interleukin-2 receptor and soluble CD8 antigen levels in serum from patients with non-resectable lung cancer Jette Vibe-Petersen 1, Niels Tvede 1, Marcus Diamant 1, Anne Arnt Kjerulff I, Hans Rahbek S0rensen 2, and Vagn Andersen 1 1Laboratoryof MedicalImmunology,Departmentof MedicineTTA, Rigshospitalet,Universityof Copenhagen,Denmark 2 Departmentof Thoracic SurgeryRT, Rigshospitalet,Universityof Copenhagen,Denmark Received 6 December 1990/Accepted 17 December 1990
Summary. In a preliminary longitudinal study two women with histologically verified adenocarcinoma of the lung, without simultaneous infectious or inflammatory conditions, were seen every 2 weeks until death. In one of the patients serum soluble interleukin-2 receptor (sIL-2R) levels rose progressively while the levels for the other patient increased during the second half of the observation period. Serum soluble CD8 antigen (sCD8 Ag) showed a pattern dissimilar to the one for sIL-2R. In a retrospective cross-sectional study circulating levels of sIL-2R and sCD8 Ag were measured before explorative thoracotomy in a total of 65 patients with histologically proven non-resectable carcinoma of the lung. The sIL-2R levels were significantly increased independently of histological subclassification while sCD8 Ag was increased only in patients with small-cell lung cancer. There was no correlation between pre-operative values and length of survival. Key words" Lung cancer - sIL-2R - Cytokines - CD8 antigen
Introduction Primary lung cancer is the most frequent malignant condition in men and occurs with increasing frequency in women. Four main histological types of bronchial carcinoma are recognized: squamous cell carcinoma (WHO l c l ) 44%, small-cell carcinoma (WHO lc2) 18%, adenocarcinoma (WHO lc3) 28% and large-cell carcinoma (WHO lc4) 10% [8]. The majority of patients with lung cancer have non-resectable tumours at the time of diagnosis and their prognosis is dismal. Except in patients with small-cell lung cancer
Offprint requests to: J. Vibe-Petersen, StenoMemorialHospital, 2 Niels Steensens Vej, DK-2820Gentofte,Denmark
systemic therapy with cytostatic agents remains experimental [22]. Therefore, new systemic therapies are badly needed. Human interleukin-2 (IL-2) is a 15-kDa glycosylated protein produced by a subset of activated T lymphocytes of helper cell phenotype. In recent clinical trials [9] adoptive transfer of IL-2 and cells with lymphokine-activated killer activity has evoked some responses in patients with melanoma, renal cell carcinoma, colon carcinoma and nonHodgkin's lymphoma. The therapeutic efficacy of this therapy in lung carcinoma has been studied in only a few patients. Those patients with advanced lung cancer responded poorly [27]. IL-2-expanded tumour-infiltrating lymphocytes, administered without systemic IL-2 to patients with lung carcinoma, gave no objective clinical responses [9]. To exert its biological effects, IL-2 must interact with a specific membrane IL-2 receptor (IL-2R) [21], which is expressed on activated normal peripheral blood mononuclear cells (PBMC: T cells, B cells, NK cells and monocytes). Using two monoclonal antibodies, anti-Tac and 7G7/B6 [17], directed against different epitopes on the Tac peptide, Rubin et al. developed an enzyme-linked immunoabsorbent assay to measure soluble IL-2R (sIL-2R; soluble Tac antigen) [16]. Activated PBMC as well as certain T and B cell lines were found to release soluble IL-2R into the culture medium [4, 7, 16]. The soluble form of the receptor (40-45 kDa) is smaller than its cellular counterpart but retains the ability to bind IL-2 [16]. Soluble Tac peptide is found in the serum of normal individuals and at elevated levels in the serum of patients with a variety of lymphoproliferative and haematological malignancies [11]. The CD8 antigen has long been used as a surface marker for the identification of cytotoxic and suppressor cells. Recently CD8-positive cells have been shown similarly to release a soluble form of the CD8 antigen (sCD8 Ag) [23], a 52-kDa homodimer, in response to actiration. The concentration of sCD8 Ag in serum has been shown to be elevated in various pediatric lymphoid malignancies [13, 14].
122 Table 1. Sequential registration of clinical parameters in two patients with pulmonary adenocarcinomaa Parameter
Experimental time (weeks) 0
2
4
65.0 -
63.5 -
62.5 -
NC Left humerus
NC
54.5
53.5
6
8
10
12
14
16
18
47.5
47.0
Patient 1 Weight (kg) Episodes of fever Chest X-ray Metastases
refused further weight control --
NC Right humerus
6 cutaneous
+
Size ?
Dead week 13
Size$'l" 2 new
Patient 2 Weight (kg) Episodes of fever Chest X-ray
59.0
Metastases
X-ray: os-~ coccygis
53.5
49.0
Dead week 19b
m
PD minor
PD PD minor single-shot irradiation
PD
NC
PD fast
PD Hepatic
NC, no change; PD, progressive disease (~>25%) b Autopsy showed pulmonary carcinoma and metastases in the lymph nodes, liver and lumbar spine
The present study was designed (a) to evaluate a sequential examination of both immunological and clinical parameters in two patients with lung cancer with one ageand sex-matched control person with blood samples drawn every 2 weeks; (b) on the basis of these data to pefform a cross-sectional examination of sIL-2R and CD8 Ag in serum samples collected before explorative thoracotomy from 65 patients with non-resectable carcinoma of the lung; (c) to estimate a possible prognostic correlation between pre-operative values of sIL-2R/sCD8 Ag and survival.
Materials and methods Study subjects. Two women with bioptically verified adenocarcinoma of the lung (WHO lc3) and one age- and sex-matched control person (respective ages 57, 60 and 56 years) were simultaneously included in this preliminaryprospective longitudinal study. The diagnosis was established by an explorative thoracotomy approximately 14 days before entry and the patients received no treatment with glucocorticoids or cytotoxic agents. They were seen every fortnight and the following clinical parameters were registered: weight, clinical examinaton, medicine consumption and episodes of fever. Every time the patient visited the out-patient clinic, a chest X-ray was taken as well as a blood sample with the following distribution: 10 ml for serum (samples stored at -80 ° C), 5 ml for plasma (Iithium heparin, stored at -80 ° C) and 5 ml for values of complete blood counts (haemoglobin, white blood cells, differential counts and platelets). The sequential evaluation of immune parameters included the following: circulating serum levels of IL-2, interferon 7 (IFN7), interleukin-6 (IL-6), tumour necrosis factor-o~ (TNFc~), a soluble or shed form of the IL-2 receptor (sIL-2R) and a soluble form of the CD8 antigen (sCD8 Ag). Informed consent from the patients, according to the Helsinki declaration, was obtained in each case. In addition, a retrospective cross-sectional examination was performed on serum samples from 65 patients with histologically proven non-resectable carcinoma of the lung. Entry criteria included no previous or concurrent malignancies as well as no previous chemotherapy/radiotherapy. The serum samples were collected before explorative thoracotomy, in the period from December 1982 until February 1988 (L. Ols-
son and H. Rahbek S0rensen), and stored at -80°C before analysis of sIL-2R and sCD8 Ag. A group of 11 age-matched healthy donors served as controls.
IL-2, IFN7, sIL-2R, sCD8 Ag and TNFo~ enzyme-linked immunosorbent assay (ELISA). IL-2 levels were determined in previously unthawed heparin-stabilised plasma while IFN7, sIL-2R, sCD8 Ag and TNFc~ levels were measured in unthawed serum samples. Fibrin was removed by incubation of plasma with CaCI2 for 2 h at 37 ° C followed by centrifugation. Plasma was diluted 1 : 2 by phosphatebuffered saline. In order to minimize proteolysis of human IFN7 in test samples, EDTA was added (10 txI EDTA/150 gl sample) for 30 min at room temperature. All parameters were measured using commercially available solidphase sandwich ELISA. The IL-2 ELISA (Intertest-2, Genzyme Corp., Boston, MA 02111, USA) was dependent on a coating antibody recognizing orte epitope on the human IL-2 molecule, which was absorbed onto polysterene microtiter plates. IL-2 present was bound, whereas umeactive components were removed by washing. An enzyme-conjugated antibody recognizing a different epitope on the human IL-2 was added and bound to solid-phase-bound IL-2. Unbound enzyme-conjugated anti-IL-2 was removed by subsequent washing, and a substrate solution was added to form a coloured product proportional to the amount of IL-2 present in the sample. The absorbance was read against a blank using a microtiter ELISA reader. Sample concentrations were determined in duplicate from a standard curve. The other assays were in principle similar to the IL-2 ELISA just described. IFN7 ELISA (Holland Biotechnology BV, Leiden) was dependent on a monoclonal antibody to human IFN7 and to a detector complex consisting of a biotinylated secondary monoclonal antibody to IFN7 and an antibiotin-aikaline-phosphatase conjugate, sIL-2R ELISA (T cell Sciences Inc., Cambridge, MA 02139, USA) was based on the binding of sIL-2R to a solid-phase-bound antibody recognizing the Tac epitope on the human IL-2R [16] and detection of the bound antigen by a horseradish-peroxidase-conjugatedantibody recognizing a different epitope on the human IL-2R (7G7/B6) [17]. sCD8 Ag ELISA (T Cell Sciences Inc., Cambridge, MA 02 139, USA) was dependent on the availability of antibody anti-CD8 mAb C9 and a second horseradish-peroxidase-conjugated anti-CD8 mAb B 12 recognizing a different epitope on the human sCD8 [23]. The TNF« Test Kit (T Cell Sciences Inc., Cambridge, MA 02 139, USA) was based on a murine mAb to human TNFc~ and a secondary horseradish-peroxidase-conjugatedmurine mAb to human TNFo«
123 • • • • Patient
No
1
---
No
2
Patient
Control
11~m
•
. . . "
9-
.
"0"
•
•
~
~
~
Upper
normal
limit
Lower
normal
limit
Upper
normal
limit
Upper
normal
limit
/
7-1
4-
3~m oo
• .i..
2-
Ex _1
10
oO
.
0 v-
•
.......
0...
• .....
..
.
"0
.....
Q•2~. ~..1
700•
°,
.
, •"
•
"0"
•-
600-
e.
x qJ m m 0.
500400-
300-
• 7._..,-..:.i....:I.:
•_.t:'«'..
--
"8.
--..
_,
Lower
normal
limit
"I"
0'
'
4'
'
8
'
1=2
'
1'6
IL-6 bioassay. IL-6 activity was determined using the IL-6-dependent mouse hybridoma cell line B 13.29 clone B9. The B9 hybridoma cells were maintained in culture medium supplemented with 5% heat-inactivated fetal calf serum (Gibco) and 20 U/tal recombinant human (th) IL-6. B9 cells used in the assay were washed three times in IL-6-free medium before culturing. Samples of 5 x 103 B9 cells were cultured together with serial dilutions of the test sample in a final volurne of 200 gl in flat-bottomed microtiter plates. After 68 h 0.5 gCi/well [3H]thymidine was added. After 5 h the cells were harvested on glassfibre filters and the [3H]thymidine incorporation was measured. All assays were performed in triplicate, and 1 U/tal IL-6 activity was defined as the concentration resulting in half-maximal [3H]thymidineincorporation. rIL-6 was used as an internal standard in all assays. Statistics. Unpaired observations were tested by the Mann-Whitney test;
paired observations by the Wilcoxon/Pratt test. The level of significance was P
