Med Oncol. & Tumor Pharmacother. VoI 9, No. 1, pp. 51-55, 1992
0736-0118/90 $3.00 + .00
B O N E M A R R O W C E L L S IN T H E D N A - S Y N T H E S I S - P H A S E IN THE MYELODYSPLASTIC SYNDROME AND LYMPHOME
A N N E - M A R I E W E N T H Z E L , 1 ANJA PORWIT, 2 KARIN L I N D A H L , 1 S U Z A N N E W I D E L L t and PETER REIZENSTEIN. 1 lHematology and 21mmunopathologylaboratories, Karolinska Hospital and Institute, Stockholm, Sweden (First submitted 8 December 1988, accepted after revision 28 November 1991.)
The bromodeoxyuridine (BRDU) labelling of bone marrow ceils was studied in 46 subjects. The labelling in 14 patients, mostly untreated, with the myelodysplastic syndrome (MDS) and four iymphoma patients was significantly (p = 0.043) higher (11.38 +_ SE 2.3 % S-phase ceils) than that of marrow cells (7.18 _+ SE 1.04%) from 14 apparently healthy normal controls and from nine patients with non hematologic disease. Six iron deficiency had numericaUy but not significantly increased values. Bone marrow samples from MDS-patients showing the highest numbers of cells in the DNA-synthesis phase had the lowest numbers of colonies and clusters in the CFU-C assay (p < 0.03). The data suggest that the DNA-synthesis period is longer in MDS than in controls. Key Words." S-phase, Bromodeoxyuridine, Myelodysplastic syndrome.
treatment protocols have been described elsewhere. 4's Four untreated patients with malignant lymphoma (2 chronic lymphatic leukemia, 1 macroglobulinemia and 1 immunocytoma with marrow infiltration) were also studied, as were 6 patients with iron deficiency anemia. 14 apparently healthy volunteers (10 women) aged 23-42 years served as controls, and nine control-patients with non-hematologic diseases (biliary cirrhosis, atrial fibrillation, pulmonary sarcoidosis seven years ago, hemochromatosis, and five patients without diagnosis, aged 39 - 84 years) were also studied. Together these 23 persons were called the 'total controls'.
INTRODUCTION
Although reliable diagnostic statistics are lacking, many experienced hematologists feel that the myelodysplastic or preleukemic syndrome (MDS) is rapidly becoming one of the most common malignant blood diseases, and one of the most refractory to treatment. 16 While the etiology - exposure to radiation or chemical mutagens is known in many cases of MDS, the pathogenesis is not. The present purpose was to examine the bone marrow cell cycle phases to see if an abnormal growth pattern in MDS can be demonstrated.
M A T E R I A L AND METHODS BrDU-labelling
The cells were Ficoll separated and washed two times in PBS (pH 7.4; KCI 0.27 g; KHzPO4 0.27 g; NazPO4 ( x HzO) 1.53 g; NaCI 10.67 g; sterile HzO up to 1000 ml). 5 x 106 cells per ml 10-4 M BrdU (5-Bromo2'-Deoxy-Uridine, Sigma) were incubated for 2 and 16 hours, respectively, in 37 ~ 5% CO2. After two washes in PBS, cytospin smears where made with the concentration of 0.5 x 106 cells per smear. The cells were fixed in ice cold acetone for at least 10 minutes and incubated for 20 minutes in 4 N HCI followed by incubation for 1 hour in 0.1% Bovine Serum Albumin 37 ~ 5% COz. The cells were then washed two times with PBS, incu-
Patients
A total of 46 patients were studied. Seventeen patients with the clinical diagnosis of the myelodysplastic syndrome were studied. After a record audit, one MDSpatient was excluded because the study preceded the diagnosis, one was already in transformation to AML, and one because he had thrombocythemia and a myeloproliferative syndrome treated with myleran. BRDUvalues were unknown to K.L., who performed the record audit. The remaining 14 patients are described in Table 1. Clinical and laboratory results of the several 51
52 A. Wenthzel et al. Table 1. Description of the patients
No.
Age (yrs)
Sex
MDS type
Transfusion req.
Duration of disease *
Previous treatment
Time between latest treatment and BRDU-test
1
80
m
I/III
yes
5
An, Cyt
> 4 weeks
2
73 (75)
m
II/II1
yes
23 (24)
Tig/Roa (P-n/Mtx/L)
> 4 weeks
3 4 5 6 7 8 9 10
73 74 75 78 78 69 76 69 (71)
m m f f f m m f
Tig/Roa Tig/Roa, Cort Tig/Roa
59
m
0
-
12
72 (74) 77 83
m f m
yes no yes no no yes yes no no no no yes
0 12 30 31 4 0 1 24 (42)
11
II II III II II III 1 I1 III I III II
10 (28) 1 > 58
(Cort, Fob P-n/Mtx/L
13 14
< 4 weeks < 4 weeks
< 4 weeks 4 weeks
* D u r a t i o n o f d i s e a s e = m o n t h s from date o f b o n e - m a r r o w d i a g n o s i s to test date. P r e v i o u s treatment; in c h r o n o l o g i c a l order.
An = Anasterone(R), Cort = Corticosteroids, Cyt = Cytosine arabinoside, fol =folic acid, L = thioguanine, mtx = methotrexate, P-n = 6-mercaptopurine, Tig = etretinate, Roa = cis-retinoic acid
Correlation of the BRD-U and CFU-C results in N D S p a t i e n t s
PBS and 0.1% NAN3. A t least 200 cells were c o u n t e d in a fluorescence microscope.
% S-phase
30
Colony assay Colony assays ( C F U - C ) were p e r f o r m e d using a s t a n d a r d d o u b l e layer t e c h n i q u e with c o m m e r c i a l l y s u p p l i e d c o n d i t i o n e d m e d i u m f r o m G i b c o in the ' F e e d e r layer'. Colonies ( m o r e than 40 cells) and clusters were c o u n t e d after seven days of incubation. T h e mean n u m b e r of colonies in the C F U - C assay of healthy donors was 65.2 (SD 32.4 ) and the m e a n n u m b e r of clusters 100.0 (SD 34.4). Normalization of the distribution of the values obtained was p e r f o r m e d by l o g a r i t h m i c transformation prior to Students t-test. S p e a r m a n p r o d u c t - m o m e n t correlation tests were also used.
~k
25
20
15
A 10
A
9
9k A 0
~k
~k
,,,,f,,,,
0
A
5
[,t,,l,,
10
15
,,l,,,,It,,,IT,J,
20
25
30
l,r,,
35
RESULTS
+trll
+0
~5
Number of colonies in CFU-C assay
Fig. 1. Colonies per 100,000 plated cells in 12 of the MDS patients in relation to the 2 hour S-phase percentages. The regression coefficient is -0.066, p < 0.03.
b a t e d with 0.1 ml a n t i - B r D U - F i t c (Becton Dickinson) 30 minutes at r o o m t e m p e r a t u r e followed by two washes in PBS a n d then m o u n t e d with 42.5% glycerine, 50%
M D S - p a t i e n t s had significantly (p = 0.047) m o r e cells in DNA-synthesis (S-) p h a s e than the normal controls, and numerically (p = 0.069) m o r e than the total controls. Patients with l y m p h o m a h a d numerically m o r e cells in S-phase than the n o r m a l (p = 0.052) and total controls (p = 0.066). Patients with any h e m a t o l o g i c a l neoplasm had significantly m o r e cells in S-phase than both the total (p = 0.033) a n d the n o r m a l (p = 0.043) controls. The n u m b e r of colonies (shown only in Fig. 1) was, as expected, significantly lower in M D S patients than in
Many marrow cells" in S-phase in M D S 53
Table 2. Percentage of bone marrow cells in DNA-synthesis (means + S.E.)
Myelodysplastic syndrome (14 patients) Patients with neoplasm of the lymphatic system (5 patients) Patients with iron deficiency anemia (6 patients) Control patients (9 patients) Volunteer controls ( 14 subjects) Total controls (23 subjects)
healthy donors (see methods, p < 0.01). Bone marrow samples with high numbers of cells in the S-phase at 2 hours showed significantly lower numbers of colonies (Fig. 1) and clusters (data not shown) than those with low DNA-synthesis (regr. coeff. = ~ . 0 6 6 , p < 0.03). No similar correlation was found for the 16 hour BRDUvalues. DISCUSSION The few lymphoma patients had mean S-phase percentages (12.9 and 17.1 at 2 and 16 hours, respectively) which were not significantly different from those in MDS (11.4 and 18.0). Similarly, the control patients with non-hematologic diseases did not have values significantly different from those in the healthy volunteers (6.3 and 10.8).This was assumed to justify the tentative amalgamation of these groups of controls vs neoplastic diseases. Earlier studies of the S-phase fraction in hematologic neoplasma have mostly dealt with 1-5 patients with " 9 %10 acute leukemm, and used mainly the tritiated thymidine method, s-H There are, however, some studies with 10-61 patients with A M L ] z15 These studies invariably show higher S-phase percentages in marrow bio9 7 1 2 15 ps~es than in aspirates.' " The present results thus have to be compared to the aspiration values. The average numbers of cells in S-phase in other studies (Table 3) vary between averages of 5.0 ~2and 10.6 l0 per cent which is lower than the present values obtained with the BRDU-technique in neoplastic disease, 11.4 and 12.9 per cent. It is not clear if the difference is secondary to methods (3-HTh vs BRDU) or to diseases (acute vs chronic neoplasma). It is conceivable that BRDU-incorporation could be influenced by treatment. However only 3/14 MDS-patients here had been given cytostatics, and this was over 4 weeks prior to the BRDU-study. The majority of the MDS-patients had received no treatment, and their values were not different from those of the treated patients. It was not possible here to find normal volunteers in the age group 60-90 years, and we could not find any
2 hours incubation
16 hours incubation
11.38-+2.3 12.9_+3.19 10.66-+ 1.19 8.56_+ 1.06 6.3-+ 1.55 7.18-+ 1.04
18.0-+2.77 17.1_+6.9 14.62-+ t .31 16.86-+2.29 10.77-+2.02 12.56-+ 1.69
earlier studies of S-phase frequencies in normal volunteers. For this reason, the patients with non-hematologic diseases, aged 39-84 years were included, essentially confirming the labelling indices in the controls. We could not determine the lineage and maturation stage of the S-phase cells. It is not known, therefore, if the increase in S-phases is caused by a change in cell composition of the bone marrow. Moreover, the iron deficiency anemia results indicate that increasing Sphase percentages cannot be excluded in non-malignant cells. It has been claimed earlier that malignantly transformed bone marrow cells have generation times longer than the normal counterparts. ~6'17The reduced formation of CFU-c in MDS-bone marrow cultures despite adequate stimulation with growth factor(s) described here confirms earlier reports, s It is thus unlikely that the S-phase in MDS has a duration of the same order of magnitude as in normal or leukemic bone marrow, in which case the present findings would suggest a shorter than normal generation time in MDS. It is not probable that the activation of a growth factor coding oncogene could explain this hypothetical rapid growth, despite a relatively hight~roduction of colony stimulating factor by MDS-cells. It is more probable that the DNA-synthesis time in MDS is prolonged more than the total generation time. This suggestion would agree with the finding of Hellstr6m et al. t9 that the amount of thymidine (not the percent of incorporating cells) incorporated into samples of MDS bone marrow is somewhat lower than that in controls. It could also explain why a high percentage of cells in S-phase is found, and why MDS is so refractory to chemotherapy. Attempts to confirm or disprove the present findings with DNA-microspectrophotometry and 3H-thymidine labelling are in progress. However, increased S-phase percentages cannot be interpreted in a simplistic fashion. It is not known why these percentages may be increased also in iron deficiency anemia. It is possible that iron deficiency also prolongs the generation time of bone marrow cells, but equally likely that there is an increased formation of red
54 A . W e n t h z e l et al. Table 3. Earlier studies of bone marrow cell percentages in the DNA synthesis (s) phase ~ % S-phase cells in marrow Ref no.
Method
7
BrdU, IV
8
3H-TdR, IV 3H-TdR in vitro
Aspiration
Biopsy
7 7.1 (4.4-11.8) 7.4 (4.1-12.6)
25
No. of patients
Diagnosis
1
AML
3
1 AML, 2 A L L
3 31 d
1 AML, 2 ALL 5 AML, 26ALL
9
3 H-TdR, IV
5.8 (5.6-6.1)
2
AML
10
3H-TdR in vitro
10.6 (5-18) 10,0 (3-18) 9.8 (2-18) 5 (2-8) 5.94 (0.1-18.8)
5
AML,CML
BrdU 3H-ara-C 12
BrdU IV
13
Flow cytometry 3H-TdR
14
Flow cytometry
15
BrdU in vitro BrdU-IV BrdU-IV
AML,CML AML,CML
6.85 (0.9-19.7) 12.0 (6.7-25.2)
2t (15-25)
10
AML
9+59 (0.4-34.0)
61 b
AML
11.58 (2.0-31.0) 15.2 (9.5-36.3)
c
AML 13 AML, 1
14
ALL
6.1 (1-12)
10
AML
6.8 (1-10) 6.8 (1-10)
10
AML
10
AML
15.9 (7-29)
a 3H-TdR=tritiated thymidine; BrdU=bromodeoxy-uridine; 3H-ara-C=tritiated cytosine arabinoside. Figures in brackets indicate ranges. IV= 5 min to 1 h infusion of BrdU or 3H-TdR; A=acute; C =chronic; M=myeloid; L=lymphatic or leukemia. A review of mainly 3H-TdR-data 1973-1980 showed median percentages between 1.5 and 13.6 in AML in a total of 511 patient aspirations. 13 b 143 samples c 12 samples d 43 samples; relapse values slightly higher (1.8-63 %) than at diagnosis
cells, which could also lead to an increase in the S-phase percentage. 5. REFERENCES 6. 1.
2.
3. 4.
Hast R, Reizenstein P, Jorpes S. Studies on human preleukemia. I. Erythroblast and iron kinetics in aregenerative anemia with hypercellular bone marrow. Scavd. J. Haematol. 1,347-354 (1987). Hast R, Beksac M, Axdorph S, Sj6gren A. M, (3st .A, Reizenstein P. Effects of retinoids on in vitro differentiation of bone marrow cells in the myelodysplastic syndrome. Med. Oncol. & Tumor Pharmacother 3, 35-38 (1986). Hast R, Reizenstein P. Sideroblastic anemia and development of leukemia. Blur 42, 203-207 (1981). Axdorph S, Beran M, Hast R, Reizenstein P. A phase I1 trial of four therapeutic regimes in MDS. 5 complete
7.
8.
9.
remissions in 22 cases (Abstract in Swedish). Ass. Sw. Phys. Nat'l Conf 1984: XLI. Hast R, Engstedt L, Jameson S, et al. Oxymetholone treatment in myelofibrosis. Blut 37, 19-26 (1987). Reizenstein P. Preleukemia. Acta Med. Scand (Editorial). 198,433-435 (1975). Raza A, Maheshwari Y, Brereton W, Preisler H D: Analysis of cell cycle characteristics and course of the disease in ANLL. Am. J Hematol. 24, 65-75 (1987). Saunders E F, Lampkin B C, Mauer A M: Variation of proliferative activity in leukemic cell populations of patients with acute leukemia. J. Clin. Investigation 46, 1356-1363 (1967). Clarkson B, Ohkita T, Ota K, Fried J. Studies of cellular proliferation in human leukemia. I. Estimation of growth rates of leukemic and normal hematopoietic cells in two adults with acute leukemia given single injections of tritiated thymidine. J. Clin Investigation 46,506-529 (1967).
M a n y m a r r o w cells in S - p h a s e in M D S
10. Raza A, Preisler H D: Double labelling of human leukemic cells using 3 H-cytarabine and monoclonal antibody against bromodeoxyuridine. Cancer Treatment Reports 697 195198 (1985). 11. Vincent P C, Borner G, Chanana A D, et al. Studies on lymphocytes. XIV. Measurement of DNA synthesis time in bovine thoracic duct lymphocytes by analysis o f labelled mitoses and by double labelling, before and after extracorporeal irradiation of the lymph. Cell Tissue Kinet 2, 235-247 (1986). 12. Raza A, Maheshwari Y, Yasin Z, Mandava N, Mayers G, Preisler H D: A new method for studying cell cycle characteristics in ANLL using double labelling with BrdU and 3HTdr. Leuk. Res. 11, 1079-1087 (1987). 13. Hiddemann W, Bfichner T, Andreeff M, W6rmann B, Melamed M R, Clarkson B D: Cell kinetics in acute leukemia. A critical reevaluation based on new data. Cancer 50, 250-258 (1982). 14. Colly L, Peters W, Hermans J, Arentsen-Honders W, Willemze R: Percentage of S-phase cells in bone marrow aspirates, biopsy specimens and bone marrow aspirates
15.
16.
17. 18.
19.
55
corrected for blood dilution from patients with acute leukemia. Leuk. Res. 11,209-2t3 (1987)~ Raza A, Ucar K, Preisler H D, Mayers G: In vitro and in vivo assessment of sensitivity to Ara-C in myeloid leukemias. Seminars in Oncol. 12 (Supp[ 3), 9-19 (1985). Sk5rberg K O, Cea R, Reizenstein P: Cellularity and cell proliferation rates in human bone marrow. Ill. Studies on bone marrow cellularity and erythrokinetics in primary and secondary polycythaemia. Acta Med. Scand. 195~ 307-3 t I (1974). Mauer A, Murphy S: Kinetic studies of cells in childhood leukemias. Am. J. Clin. Pathol. 72, 735-735 (1979). Shouten H C, Delwel R, Bot F, Hagemeijer A~ Touw I P, L6wenberg B: Characterization of clonogeneic cells in refractory anemia with excess of blasts (RAEB-CFU): Response to recombinant hematopoietic growth factors and maturation phenotypes. Leuk. Res. 137 245-251 (1989). Hellstr6m E, Robert K H, Gahrton G, et al. Therapeutic effect of low dose cytosine arabinoside, alpha interferon; l-alpha hydroxyvitamin D3 and retinoic acid in acute leukemia and myelodysplastic syndrome. Eur. J. Haematol. 40, 449-459 (1988).
0736-0118/90 $3.00 + .00
B O N E M A R R O W C E L L S IN T H E D N A - S Y N T H E S I S - P H A S E IN THE MYELODYSPLASTIC SYNDROME AND LYMPHOME
A N N E - M A R I E W E N T H Z E L , 1 ANJA PORWIT, 2 KARIN L I N D A H L , 1 S U Z A N N E W I D E L L t and PETER REIZENSTEIN. 1 lHematology and 21mmunopathologylaboratories, Karolinska Hospital and Institute, Stockholm, Sweden (First submitted 8 December 1988, accepted after revision 28 November 1991.)
The bromodeoxyuridine (BRDU) labelling of bone marrow ceils was studied in 46 subjects. The labelling in 14 patients, mostly untreated, with the myelodysplastic syndrome (MDS) and four iymphoma patients was significantly (p = 0.043) higher (11.38 +_ SE 2.3 % S-phase ceils) than that of marrow cells (7.18 _+ SE 1.04%) from 14 apparently healthy normal controls and from nine patients with non hematologic disease. Six iron deficiency had numericaUy but not significantly increased values. Bone marrow samples from MDS-patients showing the highest numbers of cells in the DNA-synthesis phase had the lowest numbers of colonies and clusters in the CFU-C assay (p < 0.03). The data suggest that the DNA-synthesis period is longer in MDS than in controls. Key Words." S-phase, Bromodeoxyuridine, Myelodysplastic syndrome.
treatment protocols have been described elsewhere. 4's Four untreated patients with malignant lymphoma (2 chronic lymphatic leukemia, 1 macroglobulinemia and 1 immunocytoma with marrow infiltration) were also studied, as were 6 patients with iron deficiency anemia. 14 apparently healthy volunteers (10 women) aged 23-42 years served as controls, and nine control-patients with non-hematologic diseases (biliary cirrhosis, atrial fibrillation, pulmonary sarcoidosis seven years ago, hemochromatosis, and five patients without diagnosis, aged 39 - 84 years) were also studied. Together these 23 persons were called the 'total controls'.
INTRODUCTION
Although reliable diagnostic statistics are lacking, many experienced hematologists feel that the myelodysplastic or preleukemic syndrome (MDS) is rapidly becoming one of the most common malignant blood diseases, and one of the most refractory to treatment. 16 While the etiology - exposure to radiation or chemical mutagens is known in many cases of MDS, the pathogenesis is not. The present purpose was to examine the bone marrow cell cycle phases to see if an abnormal growth pattern in MDS can be demonstrated.
M A T E R I A L AND METHODS BrDU-labelling
The cells were Ficoll separated and washed two times in PBS (pH 7.4; KCI 0.27 g; KHzPO4 0.27 g; NazPO4 ( x HzO) 1.53 g; NaCI 10.67 g; sterile HzO up to 1000 ml). 5 x 106 cells per ml 10-4 M BrdU (5-Bromo2'-Deoxy-Uridine, Sigma) were incubated for 2 and 16 hours, respectively, in 37 ~ 5% CO2. After two washes in PBS, cytospin smears where made with the concentration of 0.5 x 106 cells per smear. The cells were fixed in ice cold acetone for at least 10 minutes and incubated for 20 minutes in 4 N HCI followed by incubation for 1 hour in 0.1% Bovine Serum Albumin 37 ~ 5% COz. The cells were then washed two times with PBS, incu-
Patients
A total of 46 patients were studied. Seventeen patients with the clinical diagnosis of the myelodysplastic syndrome were studied. After a record audit, one MDSpatient was excluded because the study preceded the diagnosis, one was already in transformation to AML, and one because he had thrombocythemia and a myeloproliferative syndrome treated with myleran. BRDUvalues were unknown to K.L., who performed the record audit. The remaining 14 patients are described in Table 1. Clinical and laboratory results of the several 51
52 A. Wenthzel et al. Table 1. Description of the patients
No.
Age (yrs)
Sex
MDS type
Transfusion req.
Duration of disease *
Previous treatment
Time between latest treatment and BRDU-test
1
80
m
I/III
yes
5
An, Cyt
> 4 weeks
2
73 (75)
m
II/II1
yes
23 (24)
Tig/Roa (P-n/Mtx/L)
> 4 weeks
3 4 5 6 7 8 9 10
73 74 75 78 78 69 76 69 (71)
m m f f f m m f
Tig/Roa Tig/Roa, Cort Tig/Roa
59
m
0
-
12
72 (74) 77 83
m f m
yes no yes no no yes yes no no no no yes
0 12 30 31 4 0 1 24 (42)
11
II II III II II III 1 I1 III I III II
10 (28) 1 > 58
(Cort, Fob P-n/Mtx/L
13 14
< 4 weeks < 4 weeks
< 4 weeks 4 weeks
* D u r a t i o n o f d i s e a s e = m o n t h s from date o f b o n e - m a r r o w d i a g n o s i s to test date. P r e v i o u s treatment; in c h r o n o l o g i c a l order.
An = Anasterone(R), Cort = Corticosteroids, Cyt = Cytosine arabinoside, fol =folic acid, L = thioguanine, mtx = methotrexate, P-n = 6-mercaptopurine, Tig = etretinate, Roa = cis-retinoic acid
Correlation of the BRD-U and CFU-C results in N D S p a t i e n t s
PBS and 0.1% NAN3. A t least 200 cells were c o u n t e d in a fluorescence microscope.
% S-phase
30
Colony assay Colony assays ( C F U - C ) were p e r f o r m e d using a s t a n d a r d d o u b l e layer t e c h n i q u e with c o m m e r c i a l l y s u p p l i e d c o n d i t i o n e d m e d i u m f r o m G i b c o in the ' F e e d e r layer'. Colonies ( m o r e than 40 cells) and clusters were c o u n t e d after seven days of incubation. T h e mean n u m b e r of colonies in the C F U - C assay of healthy donors was 65.2 (SD 32.4 ) and the m e a n n u m b e r of clusters 100.0 (SD 34.4). Normalization of the distribution of the values obtained was p e r f o r m e d by l o g a r i t h m i c transformation prior to Students t-test. S p e a r m a n p r o d u c t - m o m e n t correlation tests were also used.
~k
25
20
15
A 10
A
9
9k A 0
~k
~k
,,,,f,,,,
0
A
5
[,t,,l,,
10
15
,,l,,,,It,,,IT,J,
20
25
30
l,r,,
35
RESULTS
+trll
+0
~5
Number of colonies in CFU-C assay
Fig. 1. Colonies per 100,000 plated cells in 12 of the MDS patients in relation to the 2 hour S-phase percentages. The regression coefficient is -0.066, p < 0.03.
b a t e d with 0.1 ml a n t i - B r D U - F i t c (Becton Dickinson) 30 minutes at r o o m t e m p e r a t u r e followed by two washes in PBS a n d then m o u n t e d with 42.5% glycerine, 50%
M D S - p a t i e n t s had significantly (p = 0.047) m o r e cells in DNA-synthesis (S-) p h a s e than the normal controls, and numerically (p = 0.069) m o r e than the total controls. Patients with l y m p h o m a h a d numerically m o r e cells in S-phase than the n o r m a l (p = 0.052) and total controls (p = 0.066). Patients with any h e m a t o l o g i c a l neoplasm had significantly m o r e cells in S-phase than both the total (p = 0.033) a n d the n o r m a l (p = 0.043) controls. The n u m b e r of colonies (shown only in Fig. 1) was, as expected, significantly lower in M D S patients than in
Many marrow cells" in S-phase in M D S 53
Table 2. Percentage of bone marrow cells in DNA-synthesis (means + S.E.)
Myelodysplastic syndrome (14 patients) Patients with neoplasm of the lymphatic system (5 patients) Patients with iron deficiency anemia (6 patients) Control patients (9 patients) Volunteer controls ( 14 subjects) Total controls (23 subjects)
healthy donors (see methods, p < 0.01). Bone marrow samples with high numbers of cells in the S-phase at 2 hours showed significantly lower numbers of colonies (Fig. 1) and clusters (data not shown) than those with low DNA-synthesis (regr. coeff. = ~ . 0 6 6 , p < 0.03). No similar correlation was found for the 16 hour BRDUvalues. DISCUSSION The few lymphoma patients had mean S-phase percentages (12.9 and 17.1 at 2 and 16 hours, respectively) which were not significantly different from those in MDS (11.4 and 18.0). Similarly, the control patients with non-hematologic diseases did not have values significantly different from those in the healthy volunteers (6.3 and 10.8).This was assumed to justify the tentative amalgamation of these groups of controls vs neoplastic diseases. Earlier studies of the S-phase fraction in hematologic neoplasma have mostly dealt with 1-5 patients with " 9 %10 acute leukemm, and used mainly the tritiated thymidine method, s-H There are, however, some studies with 10-61 patients with A M L ] z15 These studies invariably show higher S-phase percentages in marrow bio9 7 1 2 15 ps~es than in aspirates.' " The present results thus have to be compared to the aspiration values. The average numbers of cells in S-phase in other studies (Table 3) vary between averages of 5.0 ~2and 10.6 l0 per cent which is lower than the present values obtained with the BRDU-technique in neoplastic disease, 11.4 and 12.9 per cent. It is not clear if the difference is secondary to methods (3-HTh vs BRDU) or to diseases (acute vs chronic neoplasma). It is conceivable that BRDU-incorporation could be influenced by treatment. However only 3/14 MDS-patients here had been given cytostatics, and this was over 4 weeks prior to the BRDU-study. The majority of the MDS-patients had received no treatment, and their values were not different from those of the treated patients. It was not possible here to find normal volunteers in the age group 60-90 years, and we could not find any
2 hours incubation
16 hours incubation
11.38-+2.3 12.9_+3.19 10.66-+ 1.19 8.56_+ 1.06 6.3-+ 1.55 7.18-+ 1.04
18.0-+2.77 17.1_+6.9 14.62-+ t .31 16.86-+2.29 10.77-+2.02 12.56-+ 1.69
earlier studies of S-phase frequencies in normal volunteers. For this reason, the patients with non-hematologic diseases, aged 39-84 years were included, essentially confirming the labelling indices in the controls. We could not determine the lineage and maturation stage of the S-phase cells. It is not known, therefore, if the increase in S-phases is caused by a change in cell composition of the bone marrow. Moreover, the iron deficiency anemia results indicate that increasing Sphase percentages cannot be excluded in non-malignant cells. It has been claimed earlier that malignantly transformed bone marrow cells have generation times longer than the normal counterparts. ~6'17The reduced formation of CFU-c in MDS-bone marrow cultures despite adequate stimulation with growth factor(s) described here confirms earlier reports, s It is thus unlikely that the S-phase in MDS has a duration of the same order of magnitude as in normal or leukemic bone marrow, in which case the present findings would suggest a shorter than normal generation time in MDS. It is not probable that the activation of a growth factor coding oncogene could explain this hypothetical rapid growth, despite a relatively hight~roduction of colony stimulating factor by MDS-cells. It is more probable that the DNA-synthesis time in MDS is prolonged more than the total generation time. This suggestion would agree with the finding of Hellstr6m et al. t9 that the amount of thymidine (not the percent of incorporating cells) incorporated into samples of MDS bone marrow is somewhat lower than that in controls. It could also explain why a high percentage of cells in S-phase is found, and why MDS is so refractory to chemotherapy. Attempts to confirm or disprove the present findings with DNA-microspectrophotometry and 3H-thymidine labelling are in progress. However, increased S-phase percentages cannot be interpreted in a simplistic fashion. It is not known why these percentages may be increased also in iron deficiency anemia. It is possible that iron deficiency also prolongs the generation time of bone marrow cells, but equally likely that there is an increased formation of red
54 A . W e n t h z e l et al. Table 3. Earlier studies of bone marrow cell percentages in the DNA synthesis (s) phase ~ % S-phase cells in marrow Ref no.
Method
7
BrdU, IV
8
3H-TdR, IV 3H-TdR in vitro
Aspiration
Biopsy
7 7.1 (4.4-11.8) 7.4 (4.1-12.6)
25
No. of patients
Diagnosis
1
AML
3
1 AML, 2 A L L
3 31 d
1 AML, 2 ALL 5 AML, 26ALL
9
3 H-TdR, IV
5.8 (5.6-6.1)
2
AML
10
3H-TdR in vitro
10.6 (5-18) 10,0 (3-18) 9.8 (2-18) 5 (2-8) 5.94 (0.1-18.8)
5
AML,CML
BrdU 3H-ara-C 12
BrdU IV
13
Flow cytometry 3H-TdR
14
Flow cytometry
15
BrdU in vitro BrdU-IV BrdU-IV
AML,CML AML,CML
6.85 (0.9-19.7) 12.0 (6.7-25.2)
2t (15-25)
10
AML
9+59 (0.4-34.0)
61 b
AML
11.58 (2.0-31.0) 15.2 (9.5-36.3)
c
AML 13 AML, 1
14
ALL
6.1 (1-12)
10
AML
6.8 (1-10) 6.8 (1-10)
10
AML
10
AML
15.9 (7-29)
a 3H-TdR=tritiated thymidine; BrdU=bromodeoxy-uridine; 3H-ara-C=tritiated cytosine arabinoside. Figures in brackets indicate ranges. IV= 5 min to 1 h infusion of BrdU or 3H-TdR; A=acute; C =chronic; M=myeloid; L=lymphatic or leukemia. A review of mainly 3H-TdR-data 1973-1980 showed median percentages between 1.5 and 13.6 in AML in a total of 511 patient aspirations. 13 b 143 samples c 12 samples d 43 samples; relapse values slightly higher (1.8-63 %) than at diagnosis
cells, which could also lead to an increase in the S-phase percentage. 5. REFERENCES 6. 1.
2.
3. 4.
Hast R, Reizenstein P, Jorpes S. Studies on human preleukemia. I. Erythroblast and iron kinetics in aregenerative anemia with hypercellular bone marrow. Scavd. J. Haematol. 1,347-354 (1987). Hast R, Beksac M, Axdorph S, Sj6gren A. M, (3st .A, Reizenstein P. Effects of retinoids on in vitro differentiation of bone marrow cells in the myelodysplastic syndrome. Med. Oncol. & Tumor Pharmacother 3, 35-38 (1986). Hast R, Reizenstein P. Sideroblastic anemia and development of leukemia. Blur 42, 203-207 (1981). Axdorph S, Beran M, Hast R, Reizenstein P. A phase I1 trial of four therapeutic regimes in MDS. 5 complete
7.
8.
9.
remissions in 22 cases (Abstract in Swedish). Ass. Sw. Phys. Nat'l Conf 1984: XLI. Hast R, Engstedt L, Jameson S, et al. Oxymetholone treatment in myelofibrosis. Blut 37, 19-26 (1987). Reizenstein P. Preleukemia. Acta Med. Scand (Editorial). 198,433-435 (1975). Raza A, Maheshwari Y, Brereton W, Preisler H D: Analysis of cell cycle characteristics and course of the disease in ANLL. Am. J Hematol. 24, 65-75 (1987). Saunders E F, Lampkin B C, Mauer A M: Variation of proliferative activity in leukemic cell populations of patients with acute leukemia. J. Clin. Investigation 46, 1356-1363 (1967). Clarkson B, Ohkita T, Ota K, Fried J. Studies of cellular proliferation in human leukemia. I. Estimation of growth rates of leukemic and normal hematopoietic cells in two adults with acute leukemia given single injections of tritiated thymidine. J. Clin Investigation 46,506-529 (1967).
M a n y m a r r o w cells in S - p h a s e in M D S
10. Raza A, Preisler H D: Double labelling of human leukemic cells using 3 H-cytarabine and monoclonal antibody against bromodeoxyuridine. Cancer Treatment Reports 697 195198 (1985). 11. Vincent P C, Borner G, Chanana A D, et al. Studies on lymphocytes. XIV. Measurement of DNA synthesis time in bovine thoracic duct lymphocytes by analysis o f labelled mitoses and by double labelling, before and after extracorporeal irradiation of the lymph. Cell Tissue Kinet 2, 235-247 (1986). 12. Raza A, Maheshwari Y, Yasin Z, Mandava N, Mayers G, Preisler H D: A new method for studying cell cycle characteristics in ANLL using double labelling with BrdU and 3HTdr. Leuk. Res. 11, 1079-1087 (1987). 13. Hiddemann W, Bfichner T, Andreeff M, W6rmann B, Melamed M R, Clarkson B D: Cell kinetics in acute leukemia. A critical reevaluation based on new data. Cancer 50, 250-258 (1982). 14. Colly L, Peters W, Hermans J, Arentsen-Honders W, Willemze R: Percentage of S-phase cells in bone marrow aspirates, biopsy specimens and bone marrow aspirates
15.
16.
17. 18.
19.
55
corrected for blood dilution from patients with acute leukemia. Leuk. Res. 11,209-2t3 (1987)~ Raza A, Ucar K, Preisler H D, Mayers G: In vitro and in vivo assessment of sensitivity to Ara-C in myeloid leukemias. Seminars in Oncol. 12 (Supp[ 3), 9-19 (1985). Sk5rberg K O, Cea R, Reizenstein P: Cellularity and cell proliferation rates in human bone marrow. Ill. Studies on bone marrow cellularity and erythrokinetics in primary and secondary polycythaemia. Acta Med. Scand. 195~ 307-3 t I (1974). Mauer A, Murphy S: Kinetic studies of cells in childhood leukemias. Am. J. Clin. Pathol. 72, 735-735 (1979). Shouten H C, Delwel R, Bot F, Hagemeijer A~ Touw I P, L6wenberg B: Characterization of clonogeneic cells in refractory anemia with excess of blasts (RAEB-CFU): Response to recombinant hematopoietic growth factors and maturation phenotypes. Leuk. Res. 137 245-251 (1989). Hellstr6m E, Robert K H, Gahrton G, et al. Therapeutic effect of low dose cytosine arabinoside, alpha interferon; l-alpha hydroxyvitamin D3 and retinoic acid in acute leukemia and myelodysplastic syndrome. Eur. J. Haematol. 40, 449-459 (1988).
