Med Oncol. & TumorPharmacother. Vol 9 No. 4 pp. 191-197, 1992
THE
MONOCYTE PATIENTS
MASUMI
AISO,
TUMOR WITH
NECROSIS
ACUTE
0736-0118/90 $3.00 + .00
FACTOR-ALPHA
LEUKEMIA
IN COMPLETE
PRODUCTION
IN
REMISSION
YOSHIKAZU
IIZUKA, HO-IL KANG, SHIGEMASA SAWADA, TOSHITERU OHSHIMA and TAKASHI HORIE First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan
(First received 13 October 1992; accepted after revision ]3 January 1993)
Tumor necrosis factor-or (TNF-cx) production by unstimulated and lipopolysaccharide (LPS)-stimulated peripheral monocytes has been studied in 17 acute myeioid leukemia (AML) patients, 54 AML patients in complete remission (AML-CR), 9 acute lymphoblastic leukemia (ALL) patients and 13 ALL patients in complete remission (ALL-CR). TNF-oc production by the unstimulated monocytes in ALL patients (n = 6, mean: 6.6 + 4.9 u/n-d) Was higher than that of normal controls (n = 13, 0.9 5:0.7 u/ml), AML patients (n = 14, 2.0 +_2.1 u/nil) and AML-CR patients (n = 21, 1.4 + 1.2 u/ml). TNF-cc production by the LPS-stimulated monocytes of the AML-CR patients (n = 54, 12.4 + 13.4 u/ml) was significantly higher than that of the normal controls (n = 21, 3.5 _+2.5 u/ml) and the AML patients (n = 17, 2.6 _+2.4 u/ml),p < 0.01, but there were not any significant differences among the AML-CR patients and the ALL patients or the ALL-CR patients. We separated the AML-CR patients into 3 groups, depending on the length of their remission, and found that AML-CR patients with longer than 6 months (M) but less than 60 M (n = 21, 15.7 + 16.9 u/ml) and the patients with a remission longer than 60 M (n = 11, 18.2 + 15.9 u/ml) had significantly higher TNF-o~ production than that of the controls. The AML-CR patients could be separated into a high TNF value group (n = 21), in which TNF-c~ production had increased from the normal levels to higher levels than the normal or showed a sustained higher level, and a normal TNF-o~ value group (n = 13), in which TNF-o~ production had decreased from the higher level to the normal level or showed a continuously normal level. 7 out of 14 AML-CR patients in the normal group and only one out of the 21 AML-CR patients suffered a relapse. In AML-CR patients, monocyte TNF-~ production was possibly related to the prognosis of the AML-CR patients, and careful observation of the disease will be necessary as TNF-c~ production of LPS-stimulated monoeytes spontaneously decreases.
Key Words: TNF-ot, AML, ALL, Remission.
INTRODUCTION
kemia showed a spontaneously higher TNF-0~ production than n o r m a l 56 However, the relation between TNF-o~ production and the clinical stage o f the acute leukemia has not been clarified
It has been reported that in vitro production of tumor necrosis factor-~ (TNF-c0, by unstimulated peripheral monocytes (PMC) and by lipopolysaccharide (LPS)stimulated PMC obtained from cancer patients, has been found to be higher than that in the PMC of the normal controls, and a correlation between this TNF-(x production and the clinical stage o f the cancer patients) -4 Also in hematologic disorders, the leukemia cells of patients with acute myelomonocytic leukemia or acute monocytic leu-
Activation of anti-ttunor immunity is a very important factor influencing the prognosis of cancer patients, and the cytotoxicity of T N F - ~ on tumor cells is a factor in the antittnnor immunity. Therefore, it is useful to study the relationship between TNF-ct production values and the clinic',A stages in patients with acute leukemia before the start of remission induction therapy and after remission.
"To whom correspondence should be addressed: First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Ohyaguchikami-machi.Itabashi-ku,Tokyo 173, Japan
We have assessed their monocyte TNF-cc production and compared the relation with their clinical figures. 191
192 M. Aiso et al. Table 1. Patient profiles FAB 7 Group
Diagnosis
Number
Age (median)
Female/male M1 M2
M3
I
AML
17
14--68 (35)
8/9
5
2 3
AML-CR ALL
54 9
14--66 (44) 15--64 (39)
28/26 2/7
6 .
.
.
9 .
4
ALL-CR
13
14-55 (39)
7/6
.
.
.
.
21
21--41 (32)
7/14
normal control
M A T E R I A L S AND M E T H O D S
Patients' profiles. The following individuals were studied: 17 AML patients before the start of remission induction therapy (group 1); 54 AML-CR patients (group 2); 9 ALL patients before the start of remission induction therapy (group 3); 13 ALL-CR patients (group 4); and 21 normal persons (control group), Table 1. The AML patients who had been treated under short term intensive chemotherapy (DCMP-85 protocol) at our hospital from 1985 onwards, 8 had received one course of remission induction therapy and 4 courses of post remission therapy for approximately 6 months(M), and had not received any further chemotherapy unless they had a relapse. The other patients, who had been treated under pre-1985 protocols, had been given maintenance chemotherapy for three years after their remission. Therefore, the patients in this study in remission who have had more than 6 M remission hav: not been receiving any further type of chemotherapy. The ALL patients had been treated under the LVP-87 protocol for approximately 2 years, 9 and 4 of 13 ALL-CR patients had fmished their therapy regimen when we examined their TNF-oc production. Of patients receiving chemotherapy, peripheral blood samples were taken only after the adequate recovery of blood cells, so as to avoid any chemotherapeutic influence on the monocytes. Moreover, the presence of an infection and the use of steroid hormones or cytokine that might influence the monocytic functions were reasons for the exclusion of such patients from our study. Informed consent was obtained from all patients and controls prior to the start of the study.
4
M4 M5b
3
27
LI
L2
Remission duration (median)
4
1
-
-
8
4
3
6
0.1M-16Y (13M)
. .
4
9
0.1-96M (6M)
The mononuclear cells (PMC) obtained by centrifugation (400 g x 30 rain.) over Lymphocyte Separation Media (Organon Teknika Corp., Durham, NC) were washed 3 times with a RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10 % fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT). The PMC was then diluted to approximately 107 cells/ml and incubated in a human AB serum-coated plastic petri dish (35 x 10 mm; Lux, Naperville, IL) for 120 rain. at 37 ~ in a 5 % COz humidified amaosphere. After 120 min., the ~:on-adherent cells were removed by 5 gentle rinsings with a prewarmed RPMI medium with FBS, after which the adherent cells were removed by a rubber policemen. Viability of those cells was 97.1 + 0.4 % by the trypan-blue dye exclusion test. At this stage, more than 90 % of the adherent cells had ingested latex beads, and of the monocytic purity more than 95 % was assessed by using immunofluorescent staining with the Leu-M3 monoclonal antibody (Becton Dickenson, Mountain View, CA). 1~ The adherent cells (106/ml) were then cultured in a 96-well flat bottom plate (Coming, New York, NY) at 37 ~ for 24 h. in the presence or absence of lipopolysaccharide (LPS, E. Coli, 011 l:B4, Sigma, St. Louis, MO) at 20 ~tg/ml. The cell-free supematants were then removed by centrifugation at 1,200 g x 5 rain., and stored at -80 ~ until assay.
Influences of plasma on TNF-c~production. After 12 h. preincubation of adherent cells (106/ml) with plasma obtained from normal persons, AML patients or AML-CR patients, 20 t-tg of LPS was added and then cells were incubated for 24 h,. The TNF-c~ levels of cell-free supematants were assayed.
Enzyme-linked immunosorbent assay (ELISA )for TN F-o~. Cell culture conditions Ten ml of venous blood was taken from each patient and placed in glass tubes containing 500 units of heparin.
The ELISA was performed by using first and second mouse monoclonal antibodies reactive to recombinant human TNF-c~ (rH-TNF-c0, ~ kindly provided by Dr. H.
TNF-o~production in acute leukemia 193 (ABTS, Amersham Int. Plc., Amersham, UK), was added after removal of the excess antibodies, and the quantity of the reaction product was measured at 410 nm by an ELISA reader (BIO-RAD Laboratories, Hercules, CA). Triplicate wells were prepared in parallel for each study, and sensitivity of the ELISA was from 0.1 to 1130 u/ml.
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RESULTS
TNF-~ production by LPS-stimulated monocytes 0 9
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Data expressed are shown as a mean _+SD, and differences between groups were analyzed by using the unpaired Tukey's studentized range test for variable ranks, with a p value of less than 0.01 considered as statistically significant.
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Fig. 1. TNF-cc production by the LPS-stimulated monocytes in normal controls and leukemic patients. Group 1 (n = 17, mean +_ SD: 2.6 + 2.4 U/ml) untreated AML patients; group 2 (n = 54, 12.4• 13.4 U/ml) AML-CR patients; group 3 (n = 9, 7.7 + 5.2 U/ml) untreated ALL patients; and, group 4 (n = 13, 5.6 + 3.9 U/ml) ALL-CR patients. Control group (n = 21, 3.5 • 2.5 U/ml) was comprised of normal volunteers. Significant differences were noted between the control group and group 2, and between group 1 and group 2, p < 0.01. Hayashi (Technical Research Laboratory, Asahi Chemical Industry Co. Ltd., Japan). The first monoclonal antibody was immobilized overnight in a 96-well microtiter plate (Greiner Labotechnik, West Germany) at 4 ~ After 5 washings to remove the excess antibodies, standard rHTNF samples from 0.2 to 10 units/ml (Asahi Chemical Industry Co. Ltd.) and the cell-free supernatants were diluted to 5, 10, 20 and 40 % in 0.1% bovine serum albumin (GIBCO) containing PBS, pH7.4, and placed into wells and kept at 23-25 ~ for 3 h.. After 5 washings, the peroxidase-conjugated second antibody was put into the wells and kept at 23-25~ for 6 h.. Then a substrate solution for the peroxidase reaction, 2,2"-Azinobis
As can be seen in Fig. 1, TNF-~ production in the control group amounted to less than 0.1 to 7.5 u/ml (n = 21, mean: 3.5 _+2.5 u/ml), and TNF-~ production in the other groups was as follows: group 1, less than 0.1 to 8.4 u/ml (n=17, 2.6 _+ 2.4 u/ml); group 2, less than 0.1 to 76 u/ml (n = 54, 12.4 _+ 13.4 u/ml); group 3, less than 0.1 to 14.3 u/ml (n = 9, 7.7 _+5.2 u/ml); and, group 4, 0.5 to 13.2 u/ml (n = 13, 5.6 + 3.9 u/ml). Significant differences were noted between the control group and group 2, and between group 1 and 2, p < 0.01. There were no significant differences of T N F - ~ production among Mt, M2, M3, M4, and M5b FAB classifications, and their results were 13.6 _+ 8.5, 12.7 _+ 15.9, 15.3 + 14.6, 7.8 _+5.0, and 10.6 _+ 3.3 u/ml, respectively. TNF-c~ production of M4 patients was the lowest.
TNF-a production by unstimulated mono@'tes As can be seen in Fig. 2, TNF- production in the control group ranged from less than 0.I to 2.5 u/ml (n = 13, 0.9 + 0.7 u/ml), whereas TNF-~ production was as follows: group 1, less than 0.1 to 7.8 u/ml (n=14, 2.0 + 2.1 u/ml); group 2, less than 0.1 to 4.5 u/ml (n = 21, 1.4 _+ 1.2 u/rot); group 3, less than 0.1 to 11.6 u/ml (n = 6, 6.6 +4.9 u/ml); and group 4, 0.5 to 16.1 u/ml (n = 11, 3.8 _+4.2 u/ml). Significant differences were noted between the control group and group 3, p < 0.01.
Relation between 7NF-c~ production of LPS-stimulated monocytes and remission durations As TNF-c~ production of the LPS-stimulated monocytes in group 2 (AML-CR) was higher, we examined the
194 M. Aiso et al.
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relation between T N F - ~ production and remission duration. The group 2 patients were separated into 3 remission duration groupings: group 2A, for patients whose remission duration was shorter than 6 months (n = 22, median: 0.1 month); group 2B, for patients whose remission lasted 6 to 60 months (n = 21, median: 22 months); and group 2C, for patients whose remission was longer than 60 months (n = 11, median: 127 months). TNF-c~ production o f LPS-stimulated monocytes in the group 2 subgroups were as follows: in 2A, 0.3 to 23 u/ml (mean: 7.2 + 5.6); in 2B, less than 0.1 to 76 u/ml (15.7 + 16.9); and in 2C, 1.0 to 45 u/ml (18.2 _+ 15.9), Fig. 3. Significant differences were noted between the control and 2B, and between the control and 2C, p < 0.01, but no significant differences were noted between the control and 2A.
0.1
9
9
9
o
0.5 Fig. 2. TNF-o~ production by the unstimulated monocytes in normal controls and leukemic patients. Group 1 (n = 14, 2.0 + 2.1 U/ml) untreated AML patients; group 2 (n = 21, 1.4 • 1.2 U/m/) AML-CR patients; group 3 (n = 6. 6.6 • 4.9 U/ml) untreated ALL patients; and, group 4 (n = 11, 3.8 • 4.2 U/ml) ALL-CR patients. Mean TNF-o~ production of the control group (n = 13) was 0.9 + 0.7 U/m/, and a significant difference was noted between the control group and group 3, p < 0. 01.
.
~,,,
I
9
I
Fig. 3. Relationship between TNF-~ production of the LPSstimulated monocytes and the duration of remission. AML-CR patients were separated into the following three groups, according to the duration of their remission: group 2A (n = 22, 7.2 + 5.6 U/ml), remission of less than 6 months; group 2B (n = 21, 15.7 _+ 16.9 U/ml), remission of between 6 and 60 months; and group 2C (n = 11, 18.2 + 15.9 U/ml), remission longer than 60 months. The control group (n = 21) mean TNF-c~ production was 3.5 • 2.5 U/ml, and there was a significant difference between the control group and group 2B, and the control group and group 2C, p < 0.01. TNF-~ production of patients, who had a continuous remission for more than 120 months, is shown by an open circle.
TNF-oLproduction in acute leukemia 195 Table 2. Influences of plasma on TNF-ct production LPS (+) Source of monocytes LPS(-)
Plasma
Normal plasma
AML plasma
AML-CR plasma
(-)
AMLs
1.0
(1)
(2)
(1)
(2)
(3)
(1)
(2)
(3)
1.3
1.0
-
-
6.0
5.5 #
-
-
2.5#
-
5.0
3.6
-
12.2
2.0
-
8.2 #
2.0
-
0.8
2.6
2.4.
2.6
-
-
8.6
-
3.3
-
D.
-
0.4
0.8
-
-
12.0
E.
1.6
8.0
-
-
14.0
12.0
B.
C.
# autologous. Others were allogeneic. TNF-ot production of the LPS-stimulated monocytes of A M L - C R patients who had continued the remission longer than 120 months was the highest ( O in Fig 3, n = 6, mean: 20.3 _+ 16.1 u/ml).
bation with plasma. On the other hand, the same results were not shown when their monocytes were preincubated with plasma of normal persons or AML-CR patients. Those results were not different bet~veen autologous and allogeneic plasma.
The relation between relapse and fluctuations in TNF-c~ production of LPS-stimulated monocytes
DISCUSSION
TNF-c~ production in 34 of 54 AML-CR patients was assayed every 3 to 6 months, so that each patient was assayed at least twice. The A M L - C R patients were then separated into 2 groups: a high TNF value group (n = 21), in which TNF-c~ production had increased from normal levels to higher than normal levels or had shown a sustained high TNF-o~ production; and a normal TNF-c~ value group (n = 13), in which TNF-c~ production had decreased from higher than normal levels to normal levels or had shown a continuously normal T N F - a production, Fig. 4. The patients, who showed a spontaneous decrease but also increased into higher than normal levels at the next assay time, were entered into a high TNF-cc value group. Only i of 21 patients in ahigh TNF-o~ value group had a relapse, and 7 of 13 patients in a normal TNF-c~ group had a relapse. Furthermore, 5 of the 7 relapsed patients in a normal TNF-ct value group had shown a spontaneous decrease of their TNF-o~ production before their relapse, at the early stage of relapse, or just before relapse, Fig. 5. Only a relapsed patient in a high TNF value group had leukemic deposits in her skin, and she had continued a hematologic remission.
influences of plasma on TNF-o~production We could study only 5 AML-CR patients, Table 2. When the monocytes o f AML-CR patients were preincubated with plasma obtained from AML patients, their TNF-c~ production was higher than that without preincu-
In AML patients, TNF-c~ production of both unstimulated and LPS stimulated monocytes were not higher than normal levels. This result might be due to a qualitative disorder of monocytes because AML is a disorder of the committed stem cells of granulocyte-macrophages (CFUgin) ~z'~3 and/or the inhibition of monocyte functions by some factors such as leukocyte inhibitory activity (LIA).~4 When the monocytes of A M L - C R patients were preincubated with plasma of AML patients and then stimulated by LPS, TNF-c~ production was higher than that of the monocytes without preincubation. Moreover, preincubation with plasma of a normal person or a A M L - C R patient did not increase the monoc)r TNF-c~ production. These results indicated that there was not an inhibitory factor, but a stimulating factor in the AML patient's plasma, and the qualitative disorder of monocytes caused low TNF-c~ production in the AML patients. TNF-~ production by the unstimulated monocytes of ALL patients was significantly higher than that of the normal controls. As ALL is not a disorder of C-YlJ-gm different from AML, the monocyte functions of ALL patients may be normal, and their monocytes already were sufficiently stimulated by a stimulating factor in vivo and showed a high level of TNF-o~ production without LPS stimulation in vitro. For these reasons, it was possible that the monocyte TNF-c~ production of ALL patients showed no additional increase by the addition of LPS stimulation. TNF-c~ production of LPS-stimulatcd monocytes showed no significant differences between normal controis and ALL-CR patients. What was the reason for the
196 M. Aiso et al.
High TNF group
Normal TNF group
100
100
50
50
25
25
TNF u/ml 10
10
5
5
\
\ 1
i
2
3
4
5
Time of TNF assay
1
1
~
2 3 4 Time of TNF assay
Fig. 4. TNF-~ production, measured every 3 to 6 months, in 34 AML-CR patients. The results for each patient is linked by a solid bar. These AML-CI~ patients were divided into two groups: a high TNF-c~value group (n = 21) and a normal TNF-c~ value group (n = 13). TNF-o~production in the relapsed patient is shown by a closed circle (e). difference between AML-CR patients and ALL-CR patients? In pediatric patients with ALL-CR during continuous chemotherapy, it has been found that the natural killer cell cytotoxic activity is significantly lower than that in normal persons. Is According to our therapeutic protocol for ALL patients, the ALL-CR patients had received the remission maintenance therapy for about 2 years after the achievement of remission. The monocyte functions of the A L L - C R p a t i e n t s p r o b a b l y a f f e c t e d by the chemotherapeutic agents, and their monocyte TNF-ct production were not higher than the normal levels and lower than the levels of the AML-CR patients. The monocyte TNF-ct production of AML-CR patients who had a continued remission of less than 6 months showed a low level similar to the ALL-CR patients, so it was due to the same reason.
As we assayed T N F - ~ production during the clinical course of the AML-CR patients, we noted that most of the
patients in the high TNF-cc value group remained in continuous remission, and that only a patient of this group had a relapse. However, this patient also had leukemia cell deposits in her skin and her hematologic remission had continued. Therefore, it was thought that her reactions, unlike the other relapsed patients, resembled those of the cancer patients.t-4Seven of the 13 AML-CR patients in the normal TNF-ct value group had a relapse, and 5 of these 7 patients showed a spontaneous decrease of their TNF-cz production before or at the time of relapse. An activation of anti-tumor immunity is important to improve the prognosis of cancer patients, :6:7 and the cytotoxicity of TNF-ct is a factor composed with the anti-tumor immunity. From this study, it was considered that TNF-cc production of LPS-stimulated monocytes related to the prognosis of the AML-CR patients, and careful observation of AML will
TNF-o~ production in acute leukemia 197 3.
100 50 25
4.
u/ml 10
5.
TNF
6.
7. i
THE
MONOCYTE PATIENTS
MASUMI
AISO,
TUMOR WITH
NECROSIS
ACUTE
0736-0118/90 $3.00 + .00
FACTOR-ALPHA
LEUKEMIA
IN COMPLETE
PRODUCTION
IN
REMISSION
YOSHIKAZU
IIZUKA, HO-IL KANG, SHIGEMASA SAWADA, TOSHITERU OHSHIMA and TAKASHI HORIE First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan
(First received 13 October 1992; accepted after revision ]3 January 1993)
Tumor necrosis factor-or (TNF-cx) production by unstimulated and lipopolysaccharide (LPS)-stimulated peripheral monocytes has been studied in 17 acute myeioid leukemia (AML) patients, 54 AML patients in complete remission (AML-CR), 9 acute lymphoblastic leukemia (ALL) patients and 13 ALL patients in complete remission (ALL-CR). TNF-oc production by the unstimulated monocytes in ALL patients (n = 6, mean: 6.6 + 4.9 u/n-d) Was higher than that of normal controls (n = 13, 0.9 5:0.7 u/ml), AML patients (n = 14, 2.0 +_2.1 u/nil) and AML-CR patients (n = 21, 1.4 + 1.2 u/ml). TNF-cc production by the LPS-stimulated monocytes of the AML-CR patients (n = 54, 12.4 + 13.4 u/ml) was significantly higher than that of the normal controls (n = 21, 3.5 _+2.5 u/ml) and the AML patients (n = 17, 2.6 _+2.4 u/ml),p < 0.01, but there were not any significant differences among the AML-CR patients and the ALL patients or the ALL-CR patients. We separated the AML-CR patients into 3 groups, depending on the length of their remission, and found that AML-CR patients with longer than 6 months (M) but less than 60 M (n = 21, 15.7 + 16.9 u/ml) and the patients with a remission longer than 60 M (n = 11, 18.2 + 15.9 u/ml) had significantly higher TNF-o~ production than that of the controls. The AML-CR patients could be separated into a high TNF value group (n = 21), in which TNF-c~ production had increased from the normal levels to higher levels than the normal or showed a sustained higher level, and a normal TNF-o~ value group (n = 13), in which TNF-o~ production had decreased from the higher level to the normal level or showed a continuously normal level. 7 out of 14 AML-CR patients in the normal group and only one out of the 21 AML-CR patients suffered a relapse. In AML-CR patients, monocyte TNF-~ production was possibly related to the prognosis of the AML-CR patients, and careful observation of the disease will be necessary as TNF-c~ production of LPS-stimulated monoeytes spontaneously decreases.
Key Words: TNF-ot, AML, ALL, Remission.
INTRODUCTION
kemia showed a spontaneously higher TNF-0~ production than n o r m a l 56 However, the relation between TNF-o~ production and the clinical stage o f the acute leukemia has not been clarified
It has been reported that in vitro production of tumor necrosis factor-~ (TNF-c0, by unstimulated peripheral monocytes (PMC) and by lipopolysaccharide (LPS)stimulated PMC obtained from cancer patients, has been found to be higher than that in the PMC of the normal controls, and a correlation between this TNF-(x production and the clinical stage o f the cancer patients) -4 Also in hematologic disorders, the leukemia cells of patients with acute myelomonocytic leukemia or acute monocytic leu-
Activation of anti-ttunor immunity is a very important factor influencing the prognosis of cancer patients, and the cytotoxicity of T N F - ~ on tumor cells is a factor in the antittnnor immunity. Therefore, it is useful to study the relationship between TNF-ct production values and the clinic',A stages in patients with acute leukemia before the start of remission induction therapy and after remission.
"To whom correspondence should be addressed: First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Ohyaguchikami-machi.Itabashi-ku,Tokyo 173, Japan
We have assessed their monocyte TNF-cc production and compared the relation with their clinical figures. 191
192 M. Aiso et al. Table 1. Patient profiles FAB 7 Group
Diagnosis
Number
Age (median)
Female/male M1 M2
M3
I
AML
17
14--68 (35)
8/9
5
2 3
AML-CR ALL
54 9
14--66 (44) 15--64 (39)
28/26 2/7
6 .
.
.
9 .
4
ALL-CR
13
14-55 (39)
7/6
.
.
.
.
21
21--41 (32)
7/14
normal control
M A T E R I A L S AND M E T H O D S
Patients' profiles. The following individuals were studied: 17 AML patients before the start of remission induction therapy (group 1); 54 AML-CR patients (group 2); 9 ALL patients before the start of remission induction therapy (group 3); 13 ALL-CR patients (group 4); and 21 normal persons (control group), Table 1. The AML patients who had been treated under short term intensive chemotherapy (DCMP-85 protocol) at our hospital from 1985 onwards, 8 had received one course of remission induction therapy and 4 courses of post remission therapy for approximately 6 months(M), and had not received any further chemotherapy unless they had a relapse. The other patients, who had been treated under pre-1985 protocols, had been given maintenance chemotherapy for three years after their remission. Therefore, the patients in this study in remission who have had more than 6 M remission hav: not been receiving any further type of chemotherapy. The ALL patients had been treated under the LVP-87 protocol for approximately 2 years, 9 and 4 of 13 ALL-CR patients had fmished their therapy regimen when we examined their TNF-oc production. Of patients receiving chemotherapy, peripheral blood samples were taken only after the adequate recovery of blood cells, so as to avoid any chemotherapeutic influence on the monocytes. Moreover, the presence of an infection and the use of steroid hormones or cytokine that might influence the monocytic functions were reasons for the exclusion of such patients from our study. Informed consent was obtained from all patients and controls prior to the start of the study.
4
M4 M5b
3
27
LI
L2
Remission duration (median)
4
1
-
-
8
4
3
6
0.1M-16Y (13M)
. .
4
9
0.1-96M (6M)
The mononuclear cells (PMC) obtained by centrifugation (400 g x 30 rain.) over Lymphocyte Separation Media (Organon Teknika Corp., Durham, NC) were washed 3 times with a RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10 % fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT). The PMC was then diluted to approximately 107 cells/ml and incubated in a human AB serum-coated plastic petri dish (35 x 10 mm; Lux, Naperville, IL) for 120 rain. at 37 ~ in a 5 % COz humidified amaosphere. After 120 min., the ~:on-adherent cells were removed by 5 gentle rinsings with a prewarmed RPMI medium with FBS, after which the adherent cells were removed by a rubber policemen. Viability of those cells was 97.1 + 0.4 % by the trypan-blue dye exclusion test. At this stage, more than 90 % of the adherent cells had ingested latex beads, and of the monocytic purity more than 95 % was assessed by using immunofluorescent staining with the Leu-M3 monoclonal antibody (Becton Dickenson, Mountain View, CA). 1~ The adherent cells (106/ml) were then cultured in a 96-well flat bottom plate (Coming, New York, NY) at 37 ~ for 24 h. in the presence or absence of lipopolysaccharide (LPS, E. Coli, 011 l:B4, Sigma, St. Louis, MO) at 20 ~tg/ml. The cell-free supematants were then removed by centrifugation at 1,200 g x 5 rain., and stored at -80 ~ until assay.
Influences of plasma on TNF-c~production. After 12 h. preincubation of adherent cells (106/ml) with plasma obtained from normal persons, AML patients or AML-CR patients, 20 t-tg of LPS was added and then cells were incubated for 24 h,. The TNF-c~ levels of cell-free supematants were assayed.
Enzyme-linked immunosorbent assay (ELISA )for TN F-o~. Cell culture conditions Ten ml of venous blood was taken from each patient and placed in glass tubes containing 500 units of heparin.
The ELISA was performed by using first and second mouse monoclonal antibodies reactive to recombinant human TNF-c~ (rH-TNF-c0, ~ kindly provided by Dr. H.
TNF-o~production in acute leukemia 193 (ABTS, Amersham Int. Plc., Amersham, UK), was added after removal of the excess antibodies, and the quantity of the reaction product was measured at 410 nm by an ELISA reader (BIO-RAD Laboratories, Hercules, CA). Triplicate wells were prepared in parallel for each study, and sensitivity of the ELISA was from 0.1 to 1130 u/ml.
I00 5O
0 O
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Fig. 1. TNF-cc production by the LPS-stimulated monocytes in normal controls and leukemic patients. Group 1 (n = 17, mean +_ SD: 2.6 + 2.4 U/ml) untreated AML patients; group 2 (n = 54, 12.4• 13.4 U/ml) AML-CR patients; group 3 (n = 9, 7.7 + 5.2 U/ml) untreated ALL patients; and, group 4 (n = 13, 5.6 + 3.9 U/ml) ALL-CR patients. Control group (n = 21, 3.5 • 2.5 U/ml) was comprised of normal volunteers. Significant differences were noted between the control group and group 2, and between group 1 and group 2, p < 0.01. Hayashi (Technical Research Laboratory, Asahi Chemical Industry Co. Ltd., Japan). The first monoclonal antibody was immobilized overnight in a 96-well microtiter plate (Greiner Labotechnik, West Germany) at 4 ~ After 5 washings to remove the excess antibodies, standard rHTNF samples from 0.2 to 10 units/ml (Asahi Chemical Industry Co. Ltd.) and the cell-free supernatants were diluted to 5, 10, 20 and 40 % in 0.1% bovine serum albumin (GIBCO) containing PBS, pH7.4, and placed into wells and kept at 23-25 ~ for 3 h.. After 5 washings, the peroxidase-conjugated second antibody was put into the wells and kept at 23-25~ for 6 h.. Then a substrate solution for the peroxidase reaction, 2,2"-Azinobis
As can be seen in Fig. 1, TNF-~ production in the control group amounted to less than 0.1 to 7.5 u/ml (n = 21, mean: 3.5 _+2.5 u/ml), and TNF-~ production in the other groups was as follows: group 1, less than 0.1 to 8.4 u/ml (n=17, 2.6 _+ 2.4 u/ml); group 2, less than 0.1 to 76 u/ml (n = 54, 12.4 _+ 13.4 u/ml); group 3, less than 0.1 to 14.3 u/ml (n = 9, 7.7 _+5.2 u/ml); and, group 4, 0.5 to 13.2 u/ml (n = 13, 5.6 + 3.9 u/ml). Significant differences were noted between the control group and group 2, and between group 1 and 2, p < 0.01. There were no significant differences of T N F - ~ production among Mt, M2, M3, M4, and M5b FAB classifications, and their results were 13.6 _+ 8.5, 12.7 _+ 15.9, 15.3 + 14.6, 7.8 _+5.0, and 10.6 _+ 3.3 u/ml, respectively. TNF-c~ production of M4 patients was the lowest.
TNF-a production by unstimulated mono@'tes As can be seen in Fig. 2, TNF- production in the control group ranged from less than 0.I to 2.5 u/ml (n = 13, 0.9 + 0.7 u/ml), whereas TNF-~ production was as follows: group 1, less than 0.1 to 7.8 u/ml (n=14, 2.0 + 2.1 u/ml); group 2, less than 0.1 to 4.5 u/ml (n = 21, 1.4 _+ 1.2 u/rot); group 3, less than 0.1 to 11.6 u/ml (n = 6, 6.6 +4.9 u/ml); and group 4, 0.5 to 16.1 u/ml (n = 11, 3.8 _+4.2 u/ml). Significant differences were noted between the control group and group 3, p < 0.01.
Relation between 7NF-c~ production of LPS-stimulated monocytes and remission durations As TNF-c~ production of the LPS-stimulated monocytes in group 2 (AML-CR) was higher, we examined the
194 M. Aiso et al.
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relation between T N F - ~ production and remission duration. The group 2 patients were separated into 3 remission duration groupings: group 2A, for patients whose remission duration was shorter than 6 months (n = 22, median: 0.1 month); group 2B, for patients whose remission lasted 6 to 60 months (n = 21, median: 22 months); and group 2C, for patients whose remission was longer than 60 months (n = 11, median: 127 months). TNF-c~ production o f LPS-stimulated monocytes in the group 2 subgroups were as follows: in 2A, 0.3 to 23 u/ml (mean: 7.2 + 5.6); in 2B, less than 0.1 to 76 u/ml (15.7 + 16.9); and in 2C, 1.0 to 45 u/ml (18.2 _+ 15.9), Fig. 3. Significant differences were noted between the control and 2B, and between the control and 2C, p < 0.01, but no significant differences were noted between the control and 2A.
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0.5 Fig. 2. TNF-o~ production by the unstimulated monocytes in normal controls and leukemic patients. Group 1 (n = 14, 2.0 + 2.1 U/ml) untreated AML patients; group 2 (n = 21, 1.4 • 1.2 U/m/) AML-CR patients; group 3 (n = 6. 6.6 • 4.9 U/ml) untreated ALL patients; and, group 4 (n = 11, 3.8 • 4.2 U/ml) ALL-CR patients. Mean TNF-o~ production of the control group (n = 13) was 0.9 + 0.7 U/m/, and a significant difference was noted between the control group and group 3, p < 0. 01.
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Fig. 3. Relationship between TNF-~ production of the LPSstimulated monocytes and the duration of remission. AML-CR patients were separated into the following three groups, according to the duration of their remission: group 2A (n = 22, 7.2 + 5.6 U/ml), remission of less than 6 months; group 2B (n = 21, 15.7 _+ 16.9 U/ml), remission of between 6 and 60 months; and group 2C (n = 11, 18.2 + 15.9 U/ml), remission longer than 60 months. The control group (n = 21) mean TNF-c~ production was 3.5 • 2.5 U/ml, and there was a significant difference between the control group and group 2B, and the control group and group 2C, p < 0.01. TNF-~ production of patients, who had a continuous remission for more than 120 months, is shown by an open circle.
TNF-oLproduction in acute leukemia 195 Table 2. Influences of plasma on TNF-ct production LPS (+) Source of monocytes LPS(-)
Plasma
Normal plasma
AML plasma
AML-CR plasma
(-)
AMLs
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(1)
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# autologous. Others were allogeneic. TNF-ot production of the LPS-stimulated monocytes of A M L - C R patients who had continued the remission longer than 120 months was the highest ( O in Fig 3, n = 6, mean: 20.3 _+ 16.1 u/ml).
bation with plasma. On the other hand, the same results were not shown when their monocytes were preincubated with plasma of normal persons or AML-CR patients. Those results were not different bet~veen autologous and allogeneic plasma.
The relation between relapse and fluctuations in TNF-c~ production of LPS-stimulated monocytes
DISCUSSION
TNF-c~ production in 34 of 54 AML-CR patients was assayed every 3 to 6 months, so that each patient was assayed at least twice. The A M L - C R patients were then separated into 2 groups: a high TNF value group (n = 21), in which TNF-c~ production had increased from normal levels to higher than normal levels or had shown a sustained high TNF-o~ production; and a normal TNF-c~ value group (n = 13), in which TNF-c~ production had decreased from higher than normal levels to normal levels or had shown a continuously normal T N F - a production, Fig. 4. The patients, who showed a spontaneous decrease but also increased into higher than normal levels at the next assay time, were entered into a high TNF-cc value group. Only i of 21 patients in ahigh TNF-o~ value group had a relapse, and 7 of 13 patients in a normal TNF-c~ group had a relapse. Furthermore, 5 of the 7 relapsed patients in a normal TNF-ct value group had shown a spontaneous decrease of their TNF-o~ production before their relapse, at the early stage of relapse, or just before relapse, Fig. 5. Only a relapsed patient in a high TNF value group had leukemic deposits in her skin, and she had continued a hematologic remission.
influences of plasma on TNF-o~production We could study only 5 AML-CR patients, Table 2. When the monocytes o f AML-CR patients were preincubated with plasma obtained from AML patients, their TNF-c~ production was higher than that without preincu-
In AML patients, TNF-c~ production of both unstimulated and LPS stimulated monocytes were not higher than normal levels. This result might be due to a qualitative disorder of monocytes because AML is a disorder of the committed stem cells of granulocyte-macrophages (CFUgin) ~z'~3 and/or the inhibition of monocyte functions by some factors such as leukocyte inhibitory activity (LIA).~4 When the monocytes of A M L - C R patients were preincubated with plasma of AML patients and then stimulated by LPS, TNF-c~ production was higher than that of the monocytes without preincubation. Moreover, preincubation with plasma of a normal person or a A M L - C R patient did not increase the monoc)r TNF-c~ production. These results indicated that there was not an inhibitory factor, but a stimulating factor in the AML patient's plasma, and the qualitative disorder of monocytes caused low TNF-c~ production in the AML patients. TNF-~ production by the unstimulated monocytes of ALL patients was significantly higher than that of the normal controls. As ALL is not a disorder of C-YlJ-gm different from AML, the monocyte functions of ALL patients may be normal, and their monocytes already were sufficiently stimulated by a stimulating factor in vivo and showed a high level of TNF-o~ production without LPS stimulation in vitro. For these reasons, it was possible that the monocyte TNF-c~ production of ALL patients showed no additional increase by the addition of LPS stimulation. TNF-c~ production of LPS-stimulatcd monocytes showed no significant differences between normal controis and ALL-CR patients. What was the reason for the
196 M. Aiso et al.
High TNF group
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Fig. 4. TNF-~ production, measured every 3 to 6 months, in 34 AML-CR patients. The results for each patient is linked by a solid bar. These AML-CI~ patients were divided into two groups: a high TNF-c~value group (n = 21) and a normal TNF-c~ value group (n = 13). TNF-o~production in the relapsed patient is shown by a closed circle (e). difference between AML-CR patients and ALL-CR patients? In pediatric patients with ALL-CR during continuous chemotherapy, it has been found that the natural killer cell cytotoxic activity is significantly lower than that in normal persons. Is According to our therapeutic protocol for ALL patients, the ALL-CR patients had received the remission maintenance therapy for about 2 years after the achievement of remission. The monocyte functions of the A L L - C R p a t i e n t s p r o b a b l y a f f e c t e d by the chemotherapeutic agents, and their monocyte TNF-ct production were not higher than the normal levels and lower than the levels of the AML-CR patients. The monocyte TNF-ct production of AML-CR patients who had a continued remission of less than 6 months showed a low level similar to the ALL-CR patients, so it was due to the same reason.
As we assayed T N F - ~ production during the clinical course of the AML-CR patients, we noted that most of the
patients in the high TNF-cc value group remained in continuous remission, and that only a patient of this group had a relapse. However, this patient also had leukemia cell deposits in her skin and her hematologic remission had continued. Therefore, it was thought that her reactions, unlike the other relapsed patients, resembled those of the cancer patients.t-4Seven of the 13 AML-CR patients in the normal TNF-ct value group had a relapse, and 5 of these 7 patients showed a spontaneous decrease of their TNF-cz production before or at the time of relapse. An activation of anti-tumor immunity is important to improve the prognosis of cancer patients, :6:7 and the cytotoxicity of TNF-ct is a factor composed with the anti-tumor immunity. From this study, it was considered that TNF-cc production of LPS-stimulated monocytes related to the prognosis of the AML-CR patients, and careful observation of AML will
TNF-o~ production in acute leukemia 197 3.
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