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A preliminary study of polyamines in the bone-marrow plasma of adult patients with leukemia
59
Clinica Chimica Actu, 107 (1980) 59-66 @ Elsevier/North-Holland Biomedical Press
CCA 1501
A PRELIMINARY STUDY OF POLYAMINES IN THE BONE-MARROW PLASMA OF ADULT PATIENTS WITH LEUKEMIA
KENJI
NISHIOKA
a**, KOUJI
EZAKI
b and JACQUELINE
S. HART b
Departments of a Surgery/Surgical Research Laboratory, a Biochemistry and b Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute Houston, TX (U.S.A.) (Received
January
28th,
1980)
Summary Polyamines (mainly putrescine, spermidine, and spermine) whose biosynthesis is a prerequisite for cell proliferation, are potential indicators of malignant growth. To investigate the mechanism of alterations of polyamine levels in physiological fluids in human cancer, polyamine levels of bone-marrow plasma from adult patients with leukemia were studied. Significant correlations were observed between bone-marrow cellularity and spermidine, between peripheral white blood cell counts and spermidine and spermine, and between absolute blast count and spermidine and spermine among untreated patients with acute leukemia. Untreated patients with chronic leukemia showed significantly elevated levels of polyamines relative to untreated patients with acute leukemia, indicating a higher turnover of bone-marrow cells in chronic leukemia than in acute leukemia. Chemotherapy-treated patients with acute leukemia who were in remission or who did not respond to the agent showed low polyamine levels. Patients who showed a destruction of tumor cell during chemotherapy gave high levels of polyamines. Overall, these studies indicate that elevated polyamine levels are markers of cell death.
Introduction Polyamines, mainly spermidine, spermine and their precursor, recognized as having important roles in growth and proliferation
* To whom correspondence
should be addressed.
putrescine, are of cells [l-4].
60
These polyamines have been found to be elevated in serum, plasma and cerebrospinal fluid as well as in urine, of patients with a variety of tumors [1,5--111. In studying the correlation between tumor load and polyamine levels in the physiological fluids, we have chosen leukemia rather than solid tumors, since tumor load in leukemia is precisely known at a given time by cell count [ 121. Unlike other studies [ 1,5], we have also chosen bone-marrow plasma rather than urine, serum from blood, or whole bone-marrow aspirate [13,14] with the hope that polyamine levels of bone-marrow plasma would influence the polyamine levels in urine and serum, and also reflect the status of leukemia in a more sensitive manner than urine or serum due to its direct contact with the tumor cells. Thus, it is the purpose of this paper to report our preliminary findings of polyamine values on admission and during treatment of adult patients with both acute and chronic leukemia, and correlations between polyamine values and other characteristics. Materials and methods Bone-marrow aspirate was obtained in heparinized (0.5 ml containing 500 units) syringes from the posterior iliac crest of adult patients through a Rosenthal needle. For this preliminary study, 23 samples were collected from various adult patients with leukemia. Since it is difficult to obtain bone-marrow aspirates from normal volunteers, samples from patients (2 patients with carcinoma of the breast, 1 with squamous cell carcinoma, 1 with melanoma, and 1 with thrombocythemia) who had not been treated for the previous 3 months and who showed normal bone marrow without any tumor invasion by the routine histologic sections and smears, were used as a reference group. The amounts of aspirates drawn were limited to approximately 3 ml to minimize mixing with peripheral blood. The aspirate was centrifuged at 500 X g for 20 min and the supernatant was recentrifuged in the identical manner to eliminate blood cells completely from the supernatant. For calculation purposes the volume of the supernatant (bonemarrow plasma) was corrected for the volume of heparin used. A polyamine sample was prepared from the plasma fraction of the aspirate as previously described in details for the serum sample [6] to measure the total polyamines. Upon hydrolysis in 6 mol/l HCl, the polyamines were extracted from the hydrolysate by the two step procedure and analyzed by a Model 120B amino-acid analyzer. This procedure yields coefficients of variation of less than 5% for all polyamines. Since polyamine concentrations less than 0.1 nmol/ml plasma can be detected but cannot be quantified with precision, these values were expressed as detectable but not quantifiable. Results Table I shows, for the 5 patients used in the reference group, polyamine levels of the bone-marrow plasma, the clot section cellularity [12], and the peripheral white blood cell and absolute polymorphonuclear leucocyte counts. The marrow and peripheral blood values were essentially normal for all the
61 TABLE
I
POLYAMINE Patient
LEVELS
OF BONE-MARROW
Diagnosis
PLASMA
OF REFERENCE
SUBJECTS Blood
Bone marrow ~SPM a
SPD b
PTC c
ceB d
WBC
Abs. PMN e
(B) nmol/mI 1 2 3 4 5
Squamous cell carcinoma Carcinoma of breast Melanoma Thrombocythemia Carcinoma of breast
Mean S.D.
plasma
109/l
0.66 0.94 1.26 1.41 1.60
0.91 1.95 2.55 1.88 1.94
+f 0 + 0.59 0.49
20 30 15 20 25
7.1 5.7 7.2 5.2 5.3
5.7 4.0 4.2 2.8 3.8
1.17 0.38
1.85 0.59
0.22 0.30
22 5.7
6.1 0.98
4.2 1.1
a Spermine. b c d e
Spermidine. Putrescine. Bone-marrow clot section ceIhdarity. Absolute polymorphonuclear leucocyte leucocytes). f Detectable but not quantifiable.
count
(WBC
X differential
Percentage
of poIymorphonwIe=
patients. Spermidine and spermine values in the bone-marrow plasma were significantly higher than values previously obtained in our studies [6] from the blood serum of either normal donors (mean f S.D. in nmol/ml serum: spermidine, 0.33 + 0.09, t test, P < 0.0005; spermine, 0.04 + 0.02, P < 0.0005), or patients with a variety of solid tumors (spermidine, 0.39 f. 0.13, P < 0.0005; spermine, 0.037 + 0.041, P < 0.0005). Since it has been shown that the serum and plasma preparations from the identical blood contain essentially the same quantities of polyamines [ 151, this result indicates that the bone-marrow plasma contains higher amounts of polyamines than blood plasma. The bone-marrow plasma obtained from patients with acute leukemia who had not been treated were then examined, as shown in Table II. Patients were ranked according to their marrow plasma spermine values, starting with the lowest value. Although we failed to observe any significant difference in polyamine levels between the reference group and the acute leukemia patients, the following correlations were found: Spermidine vs. spermine (coefficient of correlation, 0.91); bone-marrow cellularity vs. spermidine (0.66); white blood cell (WBC) vs. spermidine (0.80) and spermine (0.89); absolute blast count (WBC X differential percentage of blast cells) vs. spermidine (0.78) and spermine (0.79). Correlations between absolute leukemic cell infiltrate (cellularity X differential percentage of blast) [12] and polyamines were also examined, but no significant correlation was observed. We then analyzed acute leukemia patients who had been treated by chemotherapy or antibiotics, as shown in Table III. The first 3 patients who were in partial (patient 1) and early complete (patient 2) remission, or who showed no response to chemotherapy (patient 3) at the time of polyamine determination had low polyamine levels. Patient 4, who was on maintenance chemotherapy, showed significantly elevated levels of spermidine (P < 0.0025) and spermine
62 TABLE II POLYAMINE LEUKEMIA Patient
LEVELS
OF BONE-MARROW
Diagnosis
PLASMA
OF UNTREATED
PATIENTS
Bone marrow SPM
SPD
Blood PTC
Cell
Abs. inf. a
(%I
(%)
nmol/ml plasma AML c AML AML AML AMML d AML AML AML
0.16 0.25 0.62 1.25 1.37 1.93 2.50 3.06
0.40 + 0.54 1.07 1.85 1.97 1.51 2.60
WITH ACUTE
WBC
Abs. blasts b
109il + + 0 + 0.42 0.54 0.77 +
90 55 30 85 90 95 95 95
80 18 13 81 86 38 48 69
2.0 2.4 2.3 4.9 13.9 19.7 33.7 76.8
0.4 1.4 0.0 2.8 12.9 7.8 11.8 55.3
a Absolute leukemic cell infiltrate (cellularity X differential percentage of myeloblast and promyelocytes). b Absolute blast count (WBC X differential percentage of myeloblasts for AML and AMML, or lymphoblasts for ALL). c Acute myelocytic leukemia. d Acute myelomonocytic leukemia.
TABLE III POLYAMINE MIA Patient
LEVELS
Diagnosis
OF BONE-MARROW
Bone marrow SPM SPD
PLASMA
OF TREATED
PATIENTS
Clinical status
Blood PTC
Cell (%)
Abs. inf. (%b)
WBC
WITH ACUTE LEUKE-
Abs. blasts
109/l I
nmol/ml plasma
-
1
AML
0.47
0.97
+
15
2
1.5
0.0
Postchem. 21 days b Partial remission
2
AML
1.31
1.66
0.54
15
2
1.0
0.0
Postchem. 10 days Remission
3
AML
1.07
1.19
0.31
100
92
3.3
2.6
Postchem. 27 days No respcmse
4
AMML
3.52
4.18
0.28
30
16
1.9
0.7
Maintenance chemotherapy
5
AMML
12.92
27.70
0.79
85
81
2.3
1.1
Postchem. 14 days Slow respcmse
6
AML
23.31
16.37
3.31
80
78
40.1
37.7
7
ALL a
8
AML
7.89
9.37
1.69
90
83
65.0
62.4
13.21
11.61
1.97
30
21
4.3
2.6
a Acute lymphocytic leukemia. b Postchemotherapy.
Postchem. 1 day Antibiotics only Antibiotics only
63 TABLE
IV
POLYAMINE Patient
LEVELS
Diag-
OF BONE-MARROW
PLASMA
OF PATIENTS
Bone marrow
WITH CHRONIC
LEUKEMIA ClinicaI status
Blood
nosis SPM -__~-
SPD
nmol/ml
plasma
PTC
Cell
Abs. inf. a
(W)
(%)
WBC
Abs. abn. b
109/I
CML c
3.20
2.23
29.50
95
11
38.9
2.7
Untreated
CLL d
8.18
4.32
1.51
70
61
78.3
41.0
Untreated
CLL
18.11
10.23
1.16
50
24
20.9
15.7
Untreated
CLL
0.85
1.17
0.41
80
53
24.3
23.6
Chemotherapy No response
CML
0.89
1.87
+
0
0
1.8
0.0
Postchem. Remission
6
CLL
1.93
2.09
0.73
10
2
5.4
1.8
Maintenance chemotherapy
I
CLL
5.69
7.03
95.22
20
1
8.3
0.7
Postchem. 10 days Remission, renal failure
a Absolute infiltrate (cellularity X differential lymphoma cells in CLL). b Absolute abnormal cells (WBC X differential cyte cells for CLL). c Chronic myelocytic leukemia. d Chronic lymphocytic leukemia.
percentage
of
myeloblasts
percentage
of myeloblasts
and promyelocytes
60 days
in CML
for CML or lymphoma-lympbo-
(P < 0.0125) relative to the reference group. Patients 5 and 6 who showed response to chemotherapy gave high polyamine levels. Patients 7 and 8, who had been on antibiotics therapy, also showed high polyamine levels. Patient 7 took carbenicillin, gentamycin and cleocin one day before the test and otherwise no treatment was given. Patient 8 had taken sumycin. Table IV depicts a study of patients with chronic leukemia. The first 3 patients, who had not been treated showed elevated levels of polyamines relative to the untreated acute leukemia group (putrescine, P < 0.05; spermidine, P < 0.0005; spermine, P < 0.0025) and to the reference group (putrescine, P < 0.1; spermidine, P < 0.05; spermine, P < 0.0125). The last 4 patients represent chemotherapy-treated chronic leukemia patients. Patients 4, 5 and 6 showed low levels of polyamines. The last patient is shown only to indicate that there may be significant elevations in polyamine values in the face of minimum tumor burden when renal failure intervenes. Discussion Based taneous in urine increased that the
on data from animal and patient studies it was proposed that sponcellular death and lysis of tumor resulted in high levels of polyamines and serum of untreated patients. Also, cell killing due to chemotherapy the release of polyamines from tumor cells. Further studies suggested spermidine excretion level may serve as a marker of tumor-cell kill,
or
64
whereas putrescine may reflect the proliferation behavior or growth fraction of tumors following chemotherapy [ 16,171. In keeping with the above hypothesis, the results obtained were examined. The low levels of polyamines exhibited by untreated patients with acute leukemia (Table II) seem to indicate that some leukemic cells turn over or lyse, but the number of cells turning over is extremely low. The first 3 treated patients who showed partial and early complete remission or no response in Table III also showed low levels of polyamines. This may be interpreted as low levels of cell destruction when the marrow-plasma polyamines were analyzed. Patients 4, 5 and 6 showed high levels of polyamines indicating that bonemarrow cells are constantly killed by maintenance chemotherapy (patient 4) and killing of bone-marrow cells by chemotherapy at the time of polyamine analysis (patients 5 and 6). Patients 7 and 8 who were treated only by antibiotics showed high levels of polyamines. This suggests possibilities which include the effect of infection stimulation on the release of polyamines from the immature cells of the marrow as has been previously reported [18] and/or the direct toxic effect of antibiotics on the immature marrow cells [19,20] and/or competitive binding of antibiotics to polyamine binding sites in the bone marrow cells [21]. Miale et al. [ 141, however, reported no significant polyamine changes by antibiotics in the total bone-marrow aspirates (bonemarrow plasma plus bone-marrow cells) of pediatric patients with acute leukemia. The reason for this difference is not clear, but the total polyamines in the bone-marrow aspirates may not change even if some bone-marrow cells are destroyed and release polyamines into the bone-marrow plasma. Untreated patients with chronic leukemia (Table IV) showed elevated polyamine levels relative to the acute leukemia group. This suggests higher cell turnover or cell death in chronic leukemia than acute leukemia. This is also in agreement with frequent elevation of serum uric acid in chronic leukemia patients [22,23]. Patients 4, 5 and 6 showed low polyamine levels indicating no substantial cell lysis due to either remission or no response to chemotherapy at the time of polyamine study. The last patient, patient 8, showed high polyamine levels due to renal failure. This finding should not be surprising since polyamines are normally excreted in the urine [ 5,151. Throughout this study, the putrescine levels were low except for one patient with chronic leukemia (Table IV) and another patient with renal failure (Table IV); therefore, it is difficult to examine the role of putrescine in our studies. However, the significant elevation of putrescine levels (P < 0.025) among treated acute leukemia patients relative to untreated patients may be reflecting regrowth activity in the bone marrow after chemotherapy, supporting the hypothesis. Judging from good correlation between spermidine and spermine, our data seem to indicate that spermine as well as spermidine can be a marker of cellkill. A correlation was found between bone-marrow cellularity and total spermidine in the bone-marrow plasma in this study. Maile et al. [14] also showed correlation between the cellularity and free polyamines in the total bonemarrow aspirates from pediatric patients with acute leukemia. This study has revealed various correlations between the marrow plasma
65
polyamine levels and the other patient characteristics in both acute and chronic leukemia. Therefore, further detailed studies concerning these correlations and the use of polyamine determinations in cell kinetic studies of leukemia patients appear promising. This procedure also may have potential as a supplemental means to characterize status of leukemia. In addition, if polyamine determinations in physiological fluids can be applied to solid tumors in a similar manner, polyamine analysis may be very effective, since it is much more difficult to characterize disease conditions in solid tumor relative to leukemia. Acknowledgements This investigation was supported by Public Health Service Grant CA 05831 from the National Cancer Institute and the Grant (No. 983) from the Kelsey and Leary Foundation. The authors are grateful to Drs. M.M. Romsdahl and S.J. Culbert for helpful discussions; Drs. G.F. Babcock and A.Y.M. Wang for critical review of the manuscript, and Miss B.J. Nowak and Mr. J.D. Whitson for their excellent technical assistance. References 1 J&me, J., PGsB, H. and Raina, A. (1978) Polyamines in rapid growth and cancer. Biochem. Biophys. Acta 473. 241-293 2 Sunkara. P.S., Rae, P.N. and Nishioka, K. (1977) Putrescine biosynthesis in mammalian cells: esse&ial for DNA synthesis but not for mitosis. Biochem. Biophys. Res. Commun. 74, 1125-1133 3 Sunkara, P.S., Rae, P.N., Nishioka, K. and Brinkley, B.R. (1979) Role of polyamines in c$tokinesis of mammalian cells. Exp. Cell Res. 119.6348 4 Sunkara, P.S., Pargac, M.B.. Nishioka, K. and Rae, P.N. (1979) Differential effects of inhibition of polyamine biosynthesis on cell cycle traverse and structure of the prematurely condensed chromosomes of normal and transformed cells. J. Cell. Physiol. 98, 451458 5 Russell, D.H. and Durie, B.G.M. (1978) Polyamines as Biochemical Markers of Normal and Malignant Growth, Raven Press, New York 6 Nishioka, K. and Romsdahl, M.M. (1974) Elevation of putrescine and spermidine in sera of patients with solid tumors. Clin. Chim. Acta 57. 156-161 7 Nishioka, K. and Romsdahl. M.M. (1977) Preliminary longitudinal studies of serum polyamines in patients with colorectal carcinoma. Cancer Lett. 3, 197-202 8 Nishioka, K., Romsdahl, M.M. and McMurtrey. M.J. (1977) Serum polyamine alterations in surgical patients with colorectal carcinoma. J. Surg. Oncol. 9, 555-562 9 Nishioka, K., Romsdahl. M.M., Fritsche, H.A. and Johnston, D.A. (1978) Polyamines in sera of patients with solid tumor. Adv. Polyamine Res. 2, 265-272 10 Nishioka, K. and Romsdahl, M.M. (1978) Polyamines as systemic biological markers of colorectal cancer. Cancer Bull. 30, 205-209 11 Takami, H., Romsdahl, M.M. and Nishioka, K. (1979) Polyamines in blood-cells as a cancer marker. Lancet 2, 912 12 Hart, J.S., Ignall. E., Trujillo, J.M., Livingston. R.B., Murphy, W., Ahearn. M. and Bodey, G.P. (1977) The clinical significance of prognostic factors in human neoplasia. In Antibiotics and Chemotherapy, Vol. 23 Fundamentals in Cancer Chemotherapy (Schabel, F.M., ed.). pp. 157-180. S. Karger. Base1 13 Rennert, 0.. Miale, T., Shukla. J., Lawson, D. and Frias, J. (1976) Polyamine concentrations in bone marrow aspirates of children with leukemia and other malignancies. Blood 47. 695-701 14 Miale, T.D., Rennert. O.M., Lawson. D.L., Shukla, J.B. and Frias, J.L. (1977) Bone marrow polyamines in children with acute leukemia as related to remission status, therapy, and cellularity of specimens. Med. Ped. Oncol. 3. 209-230 15 Russell, D.H. and Russell, S.D. (1975) Relative usefulness of measuring polyamines in serum, plasma and urine aa biochemical markers of cancer. Clin. Chem. 21, 860-863 16 Russell, D.H., Looney. W.B., Kovacs. C.J., Hopkins, H.A., Marton, L.J.. LeGendre, S.M. and Morris. H.P. (1974) Polyamine depletion of tumor tissue and subsequent elevation of spermidine in the sera of rats with 3924A hepatomas after 5-fluorouracfi administration. Cancer Res. 34. 2382-2385
66 17 18
19
20 21
22 23
Russell, D.H., Durie, B.G.M. and Salmon, S.E. (1975) Polyamines as predictors of success and failure in cancer chemotherapy. Lancet 2.797-799 Evans, W.H., Grieshaber, C.K., Miller, W.C.. Wilson, S.M. and Hoffman, H.A. (1978) Polyamine synthesis in bone marrow granulocytes: effects of cell maturity and early changes following an inflammatory stimulus. Blood 51, 1021-1029 Kancir, L.M., Tuazon, C.U., Cardella, T.A. and Sheagren. J.N. (1978) Adverse reactions to methicillin and nafcillin during treatment of serious Staphylococcus aureus infections. Arch. Int. Med. 138. 909911 Weitzman. S.A. and Stossell, T.P. (1978) Drug-induced immunological neutropenia. Lancet 1, 10681072 Kornguth, M.L.. Bayer. W.H. and Kunin, C.M. (1980) Binding of gentamicin to subcellular fractions of rabbit kidney: inhibition by spermine and other polyamines. J. Antimicrob. Chemother. 6. 121131 Spiers. A.S.D. (1977) The clinical features of chronic granulocytic leukaemia. Clin. Haematol. 6-l. 77-95 Sweet. D.L., Grolomb, H.M. and Ultmann, J.E. (1977) The clinical features of chronic lymphocytic leukaemia. Clin. Haematol. 6-l. 185-202
Clinica Chimica Actu, 107 (1980) 59-66 @ Elsevier/North-Holland Biomedical Press
CCA 1501
A PRELIMINARY STUDY OF POLYAMINES IN THE BONE-MARROW PLASMA OF ADULT PATIENTS WITH LEUKEMIA
KENJI
NISHIOKA
a**, KOUJI
EZAKI
b and JACQUELINE
S. HART b
Departments of a Surgery/Surgical Research Laboratory, a Biochemistry and b Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute Houston, TX (U.S.A.) (Received
January
28th,
1980)
Summary Polyamines (mainly putrescine, spermidine, and spermine) whose biosynthesis is a prerequisite for cell proliferation, are potential indicators of malignant growth. To investigate the mechanism of alterations of polyamine levels in physiological fluids in human cancer, polyamine levels of bone-marrow plasma from adult patients with leukemia were studied. Significant correlations were observed between bone-marrow cellularity and spermidine, between peripheral white blood cell counts and spermidine and spermine, and between absolute blast count and spermidine and spermine among untreated patients with acute leukemia. Untreated patients with chronic leukemia showed significantly elevated levels of polyamines relative to untreated patients with acute leukemia, indicating a higher turnover of bone-marrow cells in chronic leukemia than in acute leukemia. Chemotherapy-treated patients with acute leukemia who were in remission or who did not respond to the agent showed low polyamine levels. Patients who showed a destruction of tumor cell during chemotherapy gave high levels of polyamines. Overall, these studies indicate that elevated polyamine levels are markers of cell death.
Introduction Polyamines, mainly spermidine, spermine and their precursor, recognized as having important roles in growth and proliferation
* To whom correspondence
should be addressed.
putrescine, are of cells [l-4].
60
These polyamines have been found to be elevated in serum, plasma and cerebrospinal fluid as well as in urine, of patients with a variety of tumors [1,5--111. In studying the correlation between tumor load and polyamine levels in the physiological fluids, we have chosen leukemia rather than solid tumors, since tumor load in leukemia is precisely known at a given time by cell count [ 121. Unlike other studies [ 1,5], we have also chosen bone-marrow plasma rather than urine, serum from blood, or whole bone-marrow aspirate [13,14] with the hope that polyamine levels of bone-marrow plasma would influence the polyamine levels in urine and serum, and also reflect the status of leukemia in a more sensitive manner than urine or serum due to its direct contact with the tumor cells. Thus, it is the purpose of this paper to report our preliminary findings of polyamine values on admission and during treatment of adult patients with both acute and chronic leukemia, and correlations between polyamine values and other characteristics. Materials and methods Bone-marrow aspirate was obtained in heparinized (0.5 ml containing 500 units) syringes from the posterior iliac crest of adult patients through a Rosenthal needle. For this preliminary study, 23 samples were collected from various adult patients with leukemia. Since it is difficult to obtain bone-marrow aspirates from normal volunteers, samples from patients (2 patients with carcinoma of the breast, 1 with squamous cell carcinoma, 1 with melanoma, and 1 with thrombocythemia) who had not been treated for the previous 3 months and who showed normal bone marrow without any tumor invasion by the routine histologic sections and smears, were used as a reference group. The amounts of aspirates drawn were limited to approximately 3 ml to minimize mixing with peripheral blood. The aspirate was centrifuged at 500 X g for 20 min and the supernatant was recentrifuged in the identical manner to eliminate blood cells completely from the supernatant. For calculation purposes the volume of the supernatant (bonemarrow plasma) was corrected for the volume of heparin used. A polyamine sample was prepared from the plasma fraction of the aspirate as previously described in details for the serum sample [6] to measure the total polyamines. Upon hydrolysis in 6 mol/l HCl, the polyamines were extracted from the hydrolysate by the two step procedure and analyzed by a Model 120B amino-acid analyzer. This procedure yields coefficients of variation of less than 5% for all polyamines. Since polyamine concentrations less than 0.1 nmol/ml plasma can be detected but cannot be quantified with precision, these values were expressed as detectable but not quantifiable. Results Table I shows, for the 5 patients used in the reference group, polyamine levels of the bone-marrow plasma, the clot section cellularity [12], and the peripheral white blood cell and absolute polymorphonuclear leucocyte counts. The marrow and peripheral blood values were essentially normal for all the
61 TABLE
I
POLYAMINE Patient
LEVELS
OF BONE-MARROW
Diagnosis
PLASMA
OF REFERENCE
SUBJECTS Blood
Bone marrow ~SPM a
SPD b
PTC c
ceB d
WBC
Abs. PMN e
(B) nmol/mI 1 2 3 4 5
Squamous cell carcinoma Carcinoma of breast Melanoma Thrombocythemia Carcinoma of breast
Mean S.D.
plasma
109/l
0.66 0.94 1.26 1.41 1.60
0.91 1.95 2.55 1.88 1.94
+f 0 + 0.59 0.49
20 30 15 20 25
7.1 5.7 7.2 5.2 5.3
5.7 4.0 4.2 2.8 3.8
1.17 0.38
1.85 0.59
0.22 0.30
22 5.7
6.1 0.98
4.2 1.1
a Spermine. b c d e
Spermidine. Putrescine. Bone-marrow clot section ceIhdarity. Absolute polymorphonuclear leucocyte leucocytes). f Detectable but not quantifiable.
count
(WBC
X differential
Percentage
of poIymorphonwIe=
patients. Spermidine and spermine values in the bone-marrow plasma were significantly higher than values previously obtained in our studies [6] from the blood serum of either normal donors (mean f S.D. in nmol/ml serum: spermidine, 0.33 + 0.09, t test, P < 0.0005; spermine, 0.04 + 0.02, P < 0.0005), or patients with a variety of solid tumors (spermidine, 0.39 f. 0.13, P < 0.0005; spermine, 0.037 + 0.041, P < 0.0005). Since it has been shown that the serum and plasma preparations from the identical blood contain essentially the same quantities of polyamines [ 151, this result indicates that the bone-marrow plasma contains higher amounts of polyamines than blood plasma. The bone-marrow plasma obtained from patients with acute leukemia who had not been treated were then examined, as shown in Table II. Patients were ranked according to their marrow plasma spermine values, starting with the lowest value. Although we failed to observe any significant difference in polyamine levels between the reference group and the acute leukemia patients, the following correlations were found: Spermidine vs. spermine (coefficient of correlation, 0.91); bone-marrow cellularity vs. spermidine (0.66); white blood cell (WBC) vs. spermidine (0.80) and spermine (0.89); absolute blast count (WBC X differential percentage of blast cells) vs. spermidine (0.78) and spermine (0.79). Correlations between absolute leukemic cell infiltrate (cellularity X differential percentage of blast) [12] and polyamines were also examined, but no significant correlation was observed. We then analyzed acute leukemia patients who had been treated by chemotherapy or antibiotics, as shown in Table III. The first 3 patients who were in partial (patient 1) and early complete (patient 2) remission, or who showed no response to chemotherapy (patient 3) at the time of polyamine determination had low polyamine levels. Patient 4, who was on maintenance chemotherapy, showed significantly elevated levels of spermidine (P < 0.0025) and spermine
62 TABLE II POLYAMINE LEUKEMIA Patient
LEVELS
OF BONE-MARROW
Diagnosis
PLASMA
OF UNTREATED
PATIENTS
Bone marrow SPM
SPD
Blood PTC
Cell
Abs. inf. a
(%I
(%)
nmol/ml plasma AML c AML AML AML AMML d AML AML AML
0.16 0.25 0.62 1.25 1.37 1.93 2.50 3.06
0.40 + 0.54 1.07 1.85 1.97 1.51 2.60
WITH ACUTE
WBC
Abs. blasts b
109il + + 0 + 0.42 0.54 0.77 +
90 55 30 85 90 95 95 95
80 18 13 81 86 38 48 69
2.0 2.4 2.3 4.9 13.9 19.7 33.7 76.8
0.4 1.4 0.0 2.8 12.9 7.8 11.8 55.3
a Absolute leukemic cell infiltrate (cellularity X differential percentage of myeloblast and promyelocytes). b Absolute blast count (WBC X differential percentage of myeloblasts for AML and AMML, or lymphoblasts for ALL). c Acute myelocytic leukemia. d Acute myelomonocytic leukemia.
TABLE III POLYAMINE MIA Patient
LEVELS
Diagnosis
OF BONE-MARROW
Bone marrow SPM SPD
PLASMA
OF TREATED
PATIENTS
Clinical status
Blood PTC
Cell (%)
Abs. inf. (%b)
WBC
WITH ACUTE LEUKE-
Abs. blasts
109/l I
nmol/ml plasma
-
1
AML
0.47
0.97
+
15
2
1.5
0.0
Postchem. 21 days b Partial remission
2
AML
1.31
1.66
0.54
15
2
1.0
0.0
Postchem. 10 days Remission
3
AML
1.07
1.19
0.31
100
92
3.3
2.6
Postchem. 27 days No respcmse
4
AMML
3.52
4.18
0.28
30
16
1.9
0.7
Maintenance chemotherapy
5
AMML
12.92
27.70
0.79
85
81
2.3
1.1
Postchem. 14 days Slow respcmse
6
AML
23.31
16.37
3.31
80
78
40.1
37.7
7
ALL a
8
AML
7.89
9.37
1.69
90
83
65.0
62.4
13.21
11.61
1.97
30
21
4.3
2.6
a Acute lymphocytic leukemia. b Postchemotherapy.
Postchem. 1 day Antibiotics only Antibiotics only
63 TABLE
IV
POLYAMINE Patient
LEVELS
Diag-
OF BONE-MARROW
PLASMA
OF PATIENTS
Bone marrow
WITH CHRONIC
LEUKEMIA ClinicaI status
Blood
nosis SPM -__~-
SPD
nmol/ml
plasma
PTC
Cell
Abs. inf. a
(W)
(%)
WBC
Abs. abn. b
109/I
CML c
3.20
2.23
29.50
95
11
38.9
2.7
Untreated
CLL d
8.18
4.32
1.51
70
61
78.3
41.0
Untreated
CLL
18.11
10.23
1.16
50
24
20.9
15.7
Untreated
CLL
0.85
1.17
0.41
80
53
24.3
23.6
Chemotherapy No response
CML
0.89
1.87
+
0
0
1.8
0.0
Postchem. Remission
6
CLL
1.93
2.09
0.73
10
2
5.4
1.8
Maintenance chemotherapy
I
CLL
5.69
7.03
95.22
20
1
8.3
0.7
Postchem. 10 days Remission, renal failure
a Absolute infiltrate (cellularity X differential lymphoma cells in CLL). b Absolute abnormal cells (WBC X differential cyte cells for CLL). c Chronic myelocytic leukemia. d Chronic lymphocytic leukemia.
percentage
of
myeloblasts
percentage
of myeloblasts
and promyelocytes
60 days
in CML
for CML or lymphoma-lympbo-
(P < 0.0125) relative to the reference group. Patients 5 and 6 who showed response to chemotherapy gave high polyamine levels. Patients 7 and 8, who had been on antibiotics therapy, also showed high polyamine levels. Patient 7 took carbenicillin, gentamycin and cleocin one day before the test and otherwise no treatment was given. Patient 8 had taken sumycin. Table IV depicts a study of patients with chronic leukemia. The first 3 patients, who had not been treated showed elevated levels of polyamines relative to the untreated acute leukemia group (putrescine, P < 0.05; spermidine, P < 0.0005; spermine, P < 0.0025) and to the reference group (putrescine, P < 0.1; spermidine, P < 0.05; spermine, P < 0.0125). The last 4 patients represent chemotherapy-treated chronic leukemia patients. Patients 4, 5 and 6 showed low levels of polyamines. The last patient is shown only to indicate that there may be significant elevations in polyamine values in the face of minimum tumor burden when renal failure intervenes. Discussion Based taneous in urine increased that the
on data from animal and patient studies it was proposed that sponcellular death and lysis of tumor resulted in high levels of polyamines and serum of untreated patients. Also, cell killing due to chemotherapy the release of polyamines from tumor cells. Further studies suggested spermidine excretion level may serve as a marker of tumor-cell kill,
or
64
whereas putrescine may reflect the proliferation behavior or growth fraction of tumors following chemotherapy [ 16,171. In keeping with the above hypothesis, the results obtained were examined. The low levels of polyamines exhibited by untreated patients with acute leukemia (Table II) seem to indicate that some leukemic cells turn over or lyse, but the number of cells turning over is extremely low. The first 3 treated patients who showed partial and early complete remission or no response in Table III also showed low levels of polyamines. This may be interpreted as low levels of cell destruction when the marrow-plasma polyamines were analyzed. Patients 4, 5 and 6 showed high levels of polyamines indicating that bonemarrow cells are constantly killed by maintenance chemotherapy (patient 4) and killing of bone-marrow cells by chemotherapy at the time of polyamine analysis (patients 5 and 6). Patients 7 and 8 who were treated only by antibiotics showed high levels of polyamines. This suggests possibilities which include the effect of infection stimulation on the release of polyamines from the immature cells of the marrow as has been previously reported [18] and/or the direct toxic effect of antibiotics on the immature marrow cells [19,20] and/or competitive binding of antibiotics to polyamine binding sites in the bone marrow cells [21]. Miale et al. [ 141, however, reported no significant polyamine changes by antibiotics in the total bone-marrow aspirates (bonemarrow plasma plus bone-marrow cells) of pediatric patients with acute leukemia. The reason for this difference is not clear, but the total polyamines in the bone-marrow aspirates may not change even if some bone-marrow cells are destroyed and release polyamines into the bone-marrow plasma. Untreated patients with chronic leukemia (Table IV) showed elevated polyamine levels relative to the acute leukemia group. This suggests higher cell turnover or cell death in chronic leukemia than acute leukemia. This is also in agreement with frequent elevation of serum uric acid in chronic leukemia patients [22,23]. Patients 4, 5 and 6 showed low polyamine levels indicating no substantial cell lysis due to either remission or no response to chemotherapy at the time of polyamine study. The last patient, patient 8, showed high polyamine levels due to renal failure. This finding should not be surprising since polyamines are normally excreted in the urine [ 5,151. Throughout this study, the putrescine levels were low except for one patient with chronic leukemia (Table IV) and another patient with renal failure (Table IV); therefore, it is difficult to examine the role of putrescine in our studies. However, the significant elevation of putrescine levels (P < 0.025) among treated acute leukemia patients relative to untreated patients may be reflecting regrowth activity in the bone marrow after chemotherapy, supporting the hypothesis. Judging from good correlation between spermidine and spermine, our data seem to indicate that spermine as well as spermidine can be a marker of cellkill. A correlation was found between bone-marrow cellularity and total spermidine in the bone-marrow plasma in this study. Maile et al. [14] also showed correlation between the cellularity and free polyamines in the total bonemarrow aspirates from pediatric patients with acute leukemia. This study has revealed various correlations between the marrow plasma
65
polyamine levels and the other patient characteristics in both acute and chronic leukemia. Therefore, further detailed studies concerning these correlations and the use of polyamine determinations in cell kinetic studies of leukemia patients appear promising. This procedure also may have potential as a supplemental means to characterize status of leukemia. In addition, if polyamine determinations in physiological fluids can be applied to solid tumors in a similar manner, polyamine analysis may be very effective, since it is much more difficult to characterize disease conditions in solid tumor relative to leukemia. Acknowledgements This investigation was supported by Public Health Service Grant CA 05831 from the National Cancer Institute and the Grant (No. 983) from the Kelsey and Leary Foundation. The authors are grateful to Drs. M.M. Romsdahl and S.J. Culbert for helpful discussions; Drs. G.F. Babcock and A.Y.M. Wang for critical review of the manuscript, and Miss B.J. Nowak and Mr. J.D. Whitson for their excellent technical assistance. References 1 J&me, J., PGsB, H. and Raina, A. (1978) Polyamines in rapid growth and cancer. Biochem. Biophys. Acta 473. 241-293 2 Sunkara. P.S., Rae, P.N. and Nishioka, K. (1977) Putrescine biosynthesis in mammalian cells: esse&ial for DNA synthesis but not for mitosis. Biochem. Biophys. Res. Commun. 74, 1125-1133 3 Sunkara, P.S., Rae, P.N., Nishioka, K. and Brinkley, B.R. (1979) Role of polyamines in c$tokinesis of mammalian cells. Exp. Cell Res. 119.6348 4 Sunkara, P.S., Pargac, M.B.. Nishioka, K. and Rae, P.N. (1979) Differential effects of inhibition of polyamine biosynthesis on cell cycle traverse and structure of the prematurely condensed chromosomes of normal and transformed cells. J. Cell. Physiol. 98, 451458 5 Russell, D.H. and Durie, B.G.M. (1978) Polyamines as Biochemical Markers of Normal and Malignant Growth, Raven Press, New York 6 Nishioka, K. and Romsdahl, M.M. (1974) Elevation of putrescine and spermidine in sera of patients with solid tumors. Clin. Chim. Acta 57. 156-161 7 Nishioka, K. and Romsdahl. M.M. (1977) Preliminary longitudinal studies of serum polyamines in patients with colorectal carcinoma. Cancer Lett. 3, 197-202 8 Nishioka, K., Romsdahl, M.M. and McMurtrey. M.J. (1977) Serum polyamine alterations in surgical patients with colorectal carcinoma. J. Surg. Oncol. 9, 555-562 9 Nishioka, K., Romsdahl. M.M., Fritsche, H.A. and Johnston, D.A. (1978) Polyamines in sera of patients with solid tumor. Adv. Polyamine Res. 2, 265-272 10 Nishioka, K. and Romsdahl, M.M. (1978) Polyamines as systemic biological markers of colorectal cancer. Cancer Bull. 30, 205-209 11 Takami, H., Romsdahl, M.M. and Nishioka, K. (1979) Polyamines in blood-cells as a cancer marker. Lancet 2, 912 12 Hart, J.S., Ignall. E., Trujillo, J.M., Livingston. R.B., Murphy, W., Ahearn. M. and Bodey, G.P. (1977) The clinical significance of prognostic factors in human neoplasia. In Antibiotics and Chemotherapy, Vol. 23 Fundamentals in Cancer Chemotherapy (Schabel, F.M., ed.). pp. 157-180. S. Karger. Base1 13 Rennert, 0.. Miale, T., Shukla. J., Lawson, D. and Frias, J. (1976) Polyamine concentrations in bone marrow aspirates of children with leukemia and other malignancies. Blood 47. 695-701 14 Miale, T.D., Rennert. O.M., Lawson. D.L., Shukla, J.B. and Frias, J.L. (1977) Bone marrow polyamines in children with acute leukemia as related to remission status, therapy, and cellularity of specimens. Med. Ped. Oncol. 3. 209-230 15 Russell, D.H. and Russell, S.D. (1975) Relative usefulness of measuring polyamines in serum, plasma and urine aa biochemical markers of cancer. Clin. Chem. 21, 860-863 16 Russell, D.H., Looney. W.B., Kovacs. C.J., Hopkins, H.A., Marton, L.J.. LeGendre, S.M. and Morris. H.P. (1974) Polyamine depletion of tumor tissue and subsequent elevation of spermidine in the sera of rats with 3924A hepatomas after 5-fluorouracfi administration. Cancer Res. 34. 2382-2385
66 17 18
19
20 21
22 23
Russell, D.H., Durie, B.G.M. and Salmon, S.E. (1975) Polyamines as predictors of success and failure in cancer chemotherapy. Lancet 2.797-799 Evans, W.H., Grieshaber, C.K., Miller, W.C.. Wilson, S.M. and Hoffman, H.A. (1978) Polyamine synthesis in bone marrow granulocytes: effects of cell maturity and early changes following an inflammatory stimulus. Blood 51, 1021-1029 Kancir, L.M., Tuazon, C.U., Cardella, T.A. and Sheagren. J.N. (1978) Adverse reactions to methicillin and nafcillin during treatment of serious Staphylococcus aureus infections. Arch. Int. Med. 138. 909911 Weitzman. S.A. and Stossell, T.P. (1978) Drug-induced immunological neutropenia. Lancet 1, 10681072 Kornguth, M.L.. Bayer. W.H. and Kunin, C.M. (1980) Binding of gentamicin to subcellular fractions of rabbit kidney: inhibition by spermine and other polyamines. J. Antimicrob. Chemother. 6. 121131 Spiers. A.S.D. (1977) The clinical features of chronic granulocytic leukaemia. Clin. Haematol. 6-l. 77-95 Sweet. D.L., Grolomb, H.M. and Ultmann, J.E. (1977) The clinical features of chronic lymphocytic leukaemia. Clin. Haematol. 6-l. 185-202