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Elevated sister chromatid exchange and cell cycle analysis in bone marrow in childhood ALL
Elevated Sister Chromatid Exchange and Cell Cycle Analysis in Bone Marrow in Childhood ALL Nyla A. Heerema, Catherine G. Palmer, and Robert L. Baehner
ABSTRACT: Sister chromatid exchange (SCE) frequencies were analyzed in both stimulated and unstimulated bone marrow samples from eight patients recently found to have childhood acute lymphocytic leukemia (ALL). Six of the patients had elevated SCE frequencies in stimulated marrow when compared to control values. In unstimulated marrow, all four patients had elevated SCE frequencies. These data agree with peripheral leukocyte findings in ALL. Cell cycle analysis revealed no significant differences between the patient marrows and the control marrows. Since no correlation between cell cycle distribution and SCE frequency was found, it is suggested that the differences in SCE observed may be related to the type of proliferating cell. INTRODUCTION Since cytogenetic studies of cell p o p u l a t i o n s in acute l y m p h o c y t i c l e u k e m i a (ALL) of c h i l d h o o d s h o w only half the patients to have c h r o m o s o m a l damage [1-9], sister c h r o m a t i d exchange (SCE) provides an a d d i t i o n a l n e w tool for evaluating chromosome damage in ALL. Changes in SCE frequencies and patterns m a y reveal earlier or more subtle changes in the c h r o m o s o m e s than those e v i d e n c e d b y c h r o m o s o m e rearrangement. Such changes might then be of prognostic value. In previous studies from this laboratory we have d e m o n s t r a t e d that c h i l d r e n with ALL have an increased frequency of SCE in p h y t o h e m a g g l u t i n i n (PHA)-stimnlated p e r i p h e r a l blood leukocytes w h e n c o m p a r e d to age-matched control subjects [10]. The increased SCE values are present at diagnosis, before therapy, and rem a i n as long as the c h i l d r e n are on therapy. However, the SCE frequencies observed in stimulated blood leukocytes return to control levels w i t h i n 8 months after therapy has been discontinued. The distribution of SCEs, both inter- and intrachromosomal, in c h i l d r e n w i t h ALL differs from the distribution in control subjects. In the earIier studies from this laboratory we found that, although c h i l d r e n w i t h ALL had l e u k e m i a cells in their p e r i p h e r a l blood at diagnosis, we could not ascertain w h e t h e r the increased SCE occurred in l e u k e m i a cells and/or in residual normal cells. Since l e u k e m i a cells do not r e s p o n d well to PHA [11,12], and the hatFrom the Departments of Medical Genetics and Pediatrics, Indiana University School of Medicine, Indianapolis. Address requests for reprints to Dr. Nyla A. Heerema, Indiana University School of Medicine, Department of Medical Genetics, 1100 W. Michigan, Indianapolis, IN 46223. Received June 8, 1981; accepted November 6, 1981.
323 © Elsevier Science Publishing Co., Inc., 1982 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics6, 323-330 (1982) 0165-4608/82/080323-0852.75
324
Heerema et al. vest and fixation of the cultured peripheral blood cells took place at a time (96 hr) when leukemia cells should no longer be proliferating, we suspected that these responding cells may have been normal circulating lymphocytes. In order to define more precisely the cells with increased SCE, i.e., normal and/ or malignant cells, we examined SCE in bone marrow cells of children with newly diagnosed ALL. Bone marrow was studied, since it was nearly replaced by lymphoblasts. PHA-stimulated as well as unstimulated (spontaneously dividing) marrows were examined to relate the bone marrow studies to the earlier peripheral blood studies. Cell cycle analyses were included to determine if the increase we had seen might be related to a cycle of longer duration in ALL patients.
MATERIALS AND METHODS
The patients studied in this investigation were children with newly diagnosed ALL. None had received any medication. Bone marrow was aspirated for diagnostic purposes from the iliac crest into a heparinized syringe, and a portion of this marrow was used for cytogenetic studies. Two to 0.7 ml of separated lymphocytes (patients 4, 6, 7, 8, controls 1, 3, 4) or whole bone marrow (patients 1, 2, 3, 5, control 2) was cultured in medium consisting of Eagle's minimal essential medium (MEM) with 20% Gibco fetal calf serum, glutamine, nonessential amino acids, and 10 -4 M bromodeoxyuridine (BrdU). Both PHA-stimulated and unstimulated preparations were studied. All cultures were maintained at 37°C in the dark for 72 hr. Three hours before fixation, Velban (0.001 ~.g/ml) was added. After hypotonic treatment with 0.7% sodium citrate, the cells were fixed with 3:1 methanol-acetic acid fixative and dropped onto cold, wet slides. Staining for SCE distribution was accomplished by the method of Korenberg and Freedlender [13]. Twenty well-differentiated second-division metaphases were photographed, and the SCE for each subject was analyzed from projected negatives. Metaphases with fuzzy, ill-defined chromosomes were included in the analysis, as the chromosomes in ALL are notable for their poor morphology [8]. Marrows were obtained from 15 patients with newly diagnosed disease. Of these, 8 had sufficient clearly defined second-division metaphases for analysis after PHA stimulation, but only 4 had adequate spontaneously dividing metaphases in the second division. Control material consisted of bone marrow from one normal adult male, two hematologically normal former ALL patients (one male, 5 years after chemotherapy, and one female, 3 years after chemotherapy), and one 12-year-old female with a localized rhabdomyosarcoma of the eye and hematologically normal marrow. Differential staining of chromosomes that have incorporated BrdU may be used to establish whether the observed metaphases are in the first, M 1 (all darkly stained), second, M 2 (harlequin staining), or third, M 3 (approximately one-fourth darkly and three-fourths lightly stained chromatids) division in vitro. Approximately 100 metaphases per subject were analyzed in this regard. Statistical analyses were performed using the t test for paired observations for two independent samples (based on the log of the SCE values), analysis of variance for repeated measurements, and chi-square tests. RESULTS SCE in Bone Marrow of ALL Patients and Control Subjects
Clinical data at diagnosis for ALL patients in this study are shown in Table 1. One patient, with trisomy 21 Down syndrome, was the only patient not achieving complete remission and is the only patient now deceased.
325
Elevated SCE in Bone Marrow in C h i l d h o o d ALL
Table 1
Patient clinical data
Patient
Sex
Age (yr)
WBC ( × 103/mm3)
Bone marrow blasts (%)
Hemoglobin (g/dl)
Platelets ( × 103/mm3)
Cell surface markers °
1 2 3b 4 5 6 7 8
F F F M M F F M
4 3 10 8 4 2 3 12
3.4 5.2 175.0 18.7 7.0 1.8 19.7 1.0
74 50 99 86 86 80 95 91.5
9.3 5.2 9.7 12.7 8.9 8.7 5.0 11.7
138 37 56 235 272 215 15 42
N N N T N -N N
~N, Null cell; T, T cell. ~l'risomy21, deceased.
The results of the SCE studies and cell cycle analyses are s h o w n in Table 2. The SCE frequencies are calculated on a per c h r o m o s o m e basis because of a n e u p l o i d clones found in ALL, as well as the inclusion of a patient w i t h trisomy 21 and ALL in this study. I n d i v i d u a l s w i t h Down's s y n d r o m e and without l e u k e m i a p r e v i o u s l y have been s h o w n to have normal SCE frequencies [14]. In the stimulated marrows, h y p e r d i p l o i d cells were seen in four of the eight patients; in two, there was extensive aneuploidy. SCE frequencies per c h r o m o s o m e ranged from 0.177 to 0.327 for the patients, with a m e a n of 0.272 SCE per chromosome. The control SCE frequencies were 0.140-0.196, with a m e a n of 0.163 SCE per chromosome. The patient and control means differed significantly (p < 0.001), but there was some heterogeneity. W h e n the patients are c o n s i d e r e d individually, patient 8 d i d not have elevated SCE rates; and the SCE frequency for patient 7, although significantly higher than the control m e a n (p < 0.05), was m u c h lower than that of the other patients and had a value similar to that for one of the control subjects. The significance of these near-normal SCE levels is not clear. No differences in clinical findings at diagnosis were found, and the early response of these patients to t h e r a p y has been as good as that of patients w i t h elevated SCE frequencies. Their progress is being carefully m o n i t o r e d to discern any differences between these two patients and the remaining patients who d e m o n s t r a t e d elevated SCE frequencies. In the four samples with a n e u p l o i d y , there was no difference in SCE frequency in the a n e u p l o i d versus the d i p l o i d cells. A study on the distribution of SCEs in stimulated marrow s h o w e d no evidence of a b i m o d a l distribution (Fig. 1). The m e a n SCE value for unstimulated marrows was 0.189 SCE per c h r o m o s o m e for patients and 0.116 for control subjects. All the SCE frequencies of the i n d i v i d u a l u n s t i m u l a t e d patient marrows (p < 0.05), as well as the m e a n (p < 0.001), were significantly elevated w h e n c o m p a r e d to the control values. In all subjects u n s t i m u l a t e d marrows s h o w e d an SCE frequency lower than that in the PHA-stimulated marrows (p < 0.05). Analysis of variance d e m o n s t r a t e d that the differences in SCE frequency b e t w e e n control and patient values were the same in u n s t i m u l a t e d and PHA-stimulated samples.
Cell Cycle Analysis in ALL and Control Bone Marrows in Vitro The possibility that the observed SCE differences might be related to differences in the cell cycle in control and patient material was c o n s i d e r e d by c o m p a r i s o n of the frequency of cells in first, second, and third division in control subjects and leu-
326 Table 2
H e e r e m a et al.
SCE a n d cell c y c l e i n p a t i e n t s a n d c o n t r o l s a Percentage of cells in
Subject Conffols 1 2 3 4b
Aneuploidy (%)
Cuture conditions
M1
Ms
M3
0 21 42
0 78 46
0 1 12
SCE/chromosome ratio
Unstimulated 0 0 0 .
.
.
.
0 0.134 0.100
. Mean 0.116
Conffols 1 2 3 4
0 0 0 0
Stimulated
Patients 1 2 3 4 5 6 7b 8
0 15.4 0 15.0 0 28.5 . 0
Patients 1 2 3 4 5 6 7 8
0 9 56.3 30.4 14.3 10.0 0 0
30 30 33 32
65 52 33 39
5 18 34 29
0.196 0.165 0.150 0.140 Mean 0.163
0 32 0 33 51 46
0 66 0 64 46 52
0 2 0 3 3 2
0 0.169 0 0.151 0.213 0.249
Unstimulated
.
.
.
. 0
0
0
0 Mean 0.189
1 15 34 33 15 15 4 30
25 67 45 62 55 72 21 45
74 18 21 5 30 13 75 25
0.327 0.319 0.314 0.307 0.272 0.269 0.191 0.177 Mean 0.272
Stimulated
aSee text for statistical significance and P values. bNot tested--insufficient marrow.
k e m i c p a t i e n t s i n b o t h s t i m u l a t e d a n d u n s t i m u l a t e d m a r r o w s . Cell c y c l e a n a l y s i s of s t i m u l a t e d m a r r o w r e v e a l e d h e t e r o g e n e i t y i n b o t h c o n t r o l s u b j e c t s ( p < 0.005) a n d p a t i e n t s (p < 0.005). W h e n p a t i e n t s w e r e c o m p a r e d w i t h c o n t r o l s u b j e c t s , a signifi c a n t d i f f e r e n c e w a s f o u n d ( p < 0.005); c o n t r o l s h a d m o r e c e l l s i n first d i v i s i o n a n d f e w e r i n t h i r d d i v i s i o n , w h e r e a s p a t i e n t s h a d f e w e r c e l l s i n first d i v i s i o n a n d m o r e in t h i r d d i v i s i o n . T h e s e d i f f e r e n c e s , h o w e v e r , w e r e d u e to t h e c o n t r i b u t i o n of t w o p a t i e n t s (1 a n d 7) i n w h i c h 74% a n d 75% of t h e c e l l s w e r e i n t h i r d d i v i s i o n , t h e r e m a i n i n g six p a t i e n t s r e s e m b l i n g c o n t r o l s u b j e c t s i n t h e p e r c e n t a g e of c e l l s i n M 1, M s, a n d M 3. T h e d a t a o n u n s t i m u l a t e d b o n e m a r r o w of b o t h c o n t r o l s u b j e c t s a n d p a t i e n t s s h o w few cells in third division and significant differences a m o n g the control subjects.
327
Elevated SCE in Bone Marrow i n Childhood ALL 4O
30
20
m lo
30--
20
10'
n O
o.
o
o
o
o.~
o
o
o
o
.%o. %
SCEICHROMOSOME
Figure 1 SCE frequencies in individual cells of the patients ([~) and control subjects (,). Both distributions are unimodal; the patients had a much broader distribution and an elevated mean compared to the control subjects. No evidence for two populations of cells--one with control-level SCE and one with elevated SCE--was found in the patients. Dots indicate hyperdiploid cells. SCE frequencies along the entire range were found in hyperdiploid cells.
Comparison of PHA-stimulated versus u n s t i m u l a t e d control marrows showed the stimulated smaples to have significantly more cells in M 3 than the u n s t i m u l a t e d samples (p < 0.005}. In the patients there was also a significant increase in cells in M3 in stimulated as compared to u n s t i m u l a t e d samples, with a concomitant decrease in cells in M 1 (p < 0.005}. These results suggest a faster cell cycle in stimulated versus u n s t i m u l a t e d marrows.
DISCUSSION
The present study shows that an increase in SCE is found i n both stimulated and u n s t i m u l a t e d bone marrow cells of patients with n e w l y diagnosed childhood ALL, confirming the increase found previously in stimulated peripheral blood [10]. The elevated SCE frequency found i n u n s t i m u l a t e d patient marrows shows that the leukemia cells themselves show this p h e n o m e n o n . These studies on ALL are the first to demonstrate an increase in SCE in marrow cells in untreated leukemia. Previous investigations of SCE i n the marrow in other leukemias have s h o w n lower or equivalent frequencies of SCE, but none has demonstrated an increase. In diploid acute myelocytic leukemia (AML} a lower fie-
328
Heerema et al. quency of SCE in bone marrow at diagnosis as c o m p a r e d to that in control marrows or to that in remission was found [15]. A decrease in SCE frequency was demonstrated in marrow cells of untreated chronic myeloblastic l e u k e m i a (CML) patients by Becher et al. [16]. Kakati et al. [17] found no difference in SCE frequency in Ph 1positive CML but did see a significant increase in SCE frequency in two patients in blastic crisis of CML. Knuutila et al. [18] reported no change in SCE frequency in the marrow of patients with CML, AML, refractory i d i o p a t h i c sideroblastic a n e m i a (preleukemia), or malignant histiocytosis as c o m p a r e d to control marrow. However, an increase in SCE was found in marrow cells in megaloblastic a n e m i a [19]. One reason for an increase in SCE m a y be related to the duration of the cell cycle. In stimulated peripheral blood leukocytes of ALL patients, this cycle has been reported to be longer than in control cells, the majority of cells reaching second division only after 96 hr in culture [20]. This m a y result both from a delay and from decreased responses to PHA in m o n o n u c l e a r cells from c h i l d r e n w i t h ALL c o m p a r e d to healthy i n d i v i d u a l s [11,12]. The cell cycle in ALL marrow has also been reported to be slower than in normal cells [21]. In our s t u d y we have not found a d e l a y e d cell cycle in ALL marrow as c o m p a r e d to control marrow. Although the cell cycle in u n s t i m u l a t e d samples was significantly slower than that in stimulated samples, the SCE frequency was greater in the stimulated samples. Thus, we do not believe that the increase in SCE is related to a cell cycle in the ALL cells longer than that in the control cells. ALL at diagnosis and CML in blastic crisis are the only leukemic conditions in w h i c h increases in SCE bone marrow cells have been reported. This suggests a c o m m o n a l i t y in the proliferating cell related to increases in SCE. There have been previous suggestions of differences in SCE levels in different s u b p o p u l a t i o n s of normal h u m a n l y m p h o c y t e s [22-25], and there may be similarities in the types of cells involved in these disorders showing an increase in SCE. Cytomorphological and terminal d e o x y n u c l e o t i d y l transferase studies on CML patients in blastic crisis have shown that in 20% of patients the blasts have l y m p h o i d characteristics [26]. Thus blast cells of CML patients in crisis m a y be indistinguishable from blasts of children with ALL [27]. The l y m p h o c y t e descends from the p l e u r i p o t e n t stem cell w h i c h has been i m p l i c a t e d in CML [28]. These findings suggest that the elevated SCE found in c h i l d h o o d ALL m a y be characteristic of a proliferating l y m p h o c y t e . The increase in SCE we observed in PHA-stimulated cultures as c o m p a r e d to u n s t i m u l a t e d cultures also suggests an effect of PHA; i.e., w h e n different cells are stimulated, different levels of SCE are observed. This agrees with other studies in w h i c h u n s t i m u l a t e d bone marrow had lower SCE frequencies than P H A - s t i m u l a t e d l y m p h o c y t e s [18], and with the observation in our studies on ALL that no difference between stimulated marrow and stimulated peripheral blood [10] was seen. Whatever the cause of the increase in SCE in ALL, it is not exclusive to the leukemia cells, since elevated SCE values are found in stimulated p e r i p h e r a l blood as well as in stimulated and u n s t i m u l a t e d marrow. The difference in SCE frequency in control subjects and patients is the same in both stimulated and s p o n t a n e o u s l y dividing samples. Furthermore, there is no evidence for a b i m o d a l distribution of SCE in stimulated marrow in w h i c h both normal and malignant cells may be present. A n e u p l o i d y (which m a y be indicative of malignant cells) was found in four samples, but there was no difference in SCE frequency per c h r o m o s o m e in the a n e u p l o i d versus the d i p l o i d cells. This is in contrast to acute n o n l y m p h o c y t i c leukemia (ANLL) in w h i c h the a n e u p l o i d leukemic cells were found to have a low SCE frequency (SCEs per cell) as c o m p a r e d to the d i p l o i d cells with w h i c h they coexisted or the d i p l o i d cells found in remission [29]. Since SCE reflects a type of DNA damage [30-32], there may be an increase in DNA damage in ALL that occurs as part of the leukemogenic process. This is re-
Elevated SCE in Bone Marrow i n Childhood ALL
329
flected as an increase in the SCE frequency in the leukemic precursor cells. This increase in SCE in ALL patients is present at diagnosis. Patients i n a preleukemic state need to be studied to determine if the increased SCE occurs prior to the onset of clinical symptoms. However, since the earliest clinical symptoms of ALL are nonspecific, it is very difficult to identify the disease in these children. In the eight cases studied, the time from identification of the first symptoms to diagnosis was 3 days to 4 months. Serial study of patients with conditions that predispose to leukemia may help to elucidate the earliest time of SCE increase. The authors are grateful to Stuart Schwartz for expert technical assistance and to Dr. Pao-Lo Yu for aid in statistical analysis. This is publication No. 81-06 from the Department of Medical Genetics, Indiana University School of Medicine, supported by Grants USPHS T32 GM 07468, Amer. Can. In-46-T, and R01 CA 13809.
REFERENCES
1. Sandberg AA, Ishihara T, Kikuchi Y, Crosswhite LH (1964): Chromosomal differences among the acute leukemias. Ann NY Acad Sci 113,663-716. 2. Sandberg AA, Takagi N, Sofuni T, Crosswhite LH (1968): Chromosomes and causation of human cancer and leukemia. Cancer 22, 1268-1282. 3. Whang-Peng J, Freireich EJ, Oppenheim JJ, Frei E, Tjio JH (1969): Cytogenetic studies in 45 patients with acute lymphocytic leukemia. J Natl Cancer Inst 43,881-897. 4. Sandberg AA, Hossfeld D (1974): Chromosomal changes in human tumors and leukemias. In: Handbuch der Allgemeinen Pathologie, Vol. 6, W Alman, ed. Springer, Berlin, p. 141. 5. Lawler SD, Secker-Walker LM, Summersgill BM, Reeves BR, Lewis J, Kay H, Hardisty RM (1975): Chromosome banding studies in acute leukemia at diagnosis. Scand J Haematol 15, 312-320. 6. Whang-Peng J, Knutsen T, Ziegler J, Leventhal B (1976): Cytogenetic studies in acute lymphocytic leukemia--Special emphasis on long-term survival Med Pediatr Oncol 2, 333-351. 7. Zuelzer WW, Inoue S, Thompson RE, Ottenbreit MJ (1976): Long-term cytogenetic studies in acute leukemia of children: The nature of relapse. Am J Hematol 1,143-190. 8. Oshimura M, Freeman AI, Sandberg AA (1977): Chromosomes and causation of human cancer and leukemia. XXVI. Banding studies in acute lymphoblastic leukemia (ALL). Cancer 40, 1161-1172. 9. Swansbury GJ, Secker-Walker LM, Lawler SD, Hardisty RM, Sallan SE, Garson OM, Sakurai M (1981): Chromosomal findings in acute lymphoblastic leukaemia of childhood: An independent prognostic factor Lancet 2, 249-250. 10. Otter M, Palmer CG, Baehner RL (1979): Sister chromatid exchange in lymphocytes from patients with acute lymphoblastic leukemia. Hum Genet 52, 185-192. 11. Fairchild SS, Cohen JJ (1979): Delayed Initogenic response to phytohemagglutinin in acute lymphoblastic leukemia. Leuk Res 3, 163-170. 12. Melief, CJM, Schweitzer M, Zeylemaker WP, Verhagen EH, Eijsvoogel VP (1973): Some immunological properties of lymphoid cells from patients with acute lymphocytic leukemia (ALL). Clin Exp Immunol 15,131-143. 13. Korenberg JR, Freedlender EF (1974): Giemsa technique for the detection of sister chromatid exchanges. Chromosoma 48, 355-360. 14. Yu, CW, Borgaonkar DS (1977): Normal rate of sister chromatid exchange in Down syndrome Clin Genet 11,397-401. 15. Abe S, Kakati S, Sandberg AA (1979): Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in acute myeloblastic leukemia (AML). Cancer Genet Cytogenet 1, 115-130. 16. Becher R, Schmidt CG, Theis G, Hossfeld DK (1979): Sister chromatid exchange in Ph 1positive chronic myelocytic leukemia. Int J Cancer 24, 713-716.
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17. Kakati S, Abe S, Sandberg AA (1978): Sister chromatid exchange in Philadelphia chromosome (Phl)-positive leukemia. Cancer Res 38, 2918-2921. 18. Knuutila S, Helminen E, Vuipio P, de la Chapelle A (1978): Sister chromatid exchanges in human bone marrow cells. I. Control subjects and patients with leukemia. Hereditas 88, 189-196. 19. Knuutila S, Helminen E, Vuopio P, de la Chapelle A (1978): Increased sister chromatid exchange in megaloblastic anemia--Studies on bone marrow cells and lymphocytes. Hereditas 89, 175-181. 20. Otter M (1979): Sister chromatid exchange in lymphocytes from patients with acute lymphoblastic leukemia. Ph.D. Thesis, Indiana University. 21. Killmann SA (1968): Acute leukemia: The kinetics of leukemic blast cells in m a n - - A n analytical review. Ser Haematol 1, 38-102. 22. Snope AJ and Rary JM (1979): Cell-cycle duration and sister-chromatid exchange frequency in cultured human lymphocytes. Mutat Res 63, 345-349. 23. Santesson B, Lindahl-Kiessling K, Mattsson A (1979): SCE in B and T lymphocytes: Possible implications for Bloom's syndrome. Clin Genet 16, 133-135. 24. Lindblad A and Lambert B (1981): Relation between sister chromatid exchange, cell proliferation and proportion of B and T cells in human lymphocyte cultures. Hum Genet 57, 31-34. 25. Ockey CH (1980): Differences between "spontaneous" and induced sister-chromatid exchanges with fixation time and their chromosome localization. Cytogenet Cell Genet 26, 223-235. 26. Marks SM, Baltimore D, McCaffrey R (1978): Terminal transferase as a predictor of initial responsiveness to vincristine and prednisone in blastic chronic myelogenous leukemia. New Engl J Med 298, 812-814. 27. Rosenthal S, Canellos GP, Whang-Peng J, Gralnick HR (1977): Blast crisis of chronic granulocytic leukemia: Morphologic variants and therapeutic implications. Am J Med 63, 542-547. 28. Fialkow PJ, Denman AM, Singer J, Jacobson RJ, Lowenthal MN (1978): Human myeloproliferative disorders: Clonal origin in pluripotent stem cells. In: Clarkson B, et al, eds., Differentiation of Normal and Neoplastic Hematopoietic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 131-144. 29. Abe S, Sandberg AA (1980): Sister chromatid exchange and growth kinetics of marrow cells in anenploid acute nonlymphocytic leukemias. Cancer Res 40, 1292-1299. 30. Latt SA (1974): Sister chromatid exchanges, indices of human chromosome damage and repair: Detection by fluorescence and induction by mitomycin C. Proc Natl Acad Sci USA 71, 3162-3166. 31. Latt SA, Schreck RR, Loveday KS, Dougherty CP, Shuler CF (1980): Sister chromatid exchanges. Adv Hum Genet 10, 267-331. 32. Gebbart E (1981): Sister chromatid exchange (SCE) and structural aberration in mutagenicity testing. Hum Genet 58, 235-254.
ABSTRACT: Sister chromatid exchange (SCE) frequencies were analyzed in both stimulated and unstimulated bone marrow samples from eight patients recently found to have childhood acute lymphocytic leukemia (ALL). Six of the patients had elevated SCE frequencies in stimulated marrow when compared to control values. In unstimulated marrow, all four patients had elevated SCE frequencies. These data agree with peripheral leukocyte findings in ALL. Cell cycle analysis revealed no significant differences between the patient marrows and the control marrows. Since no correlation between cell cycle distribution and SCE frequency was found, it is suggested that the differences in SCE observed may be related to the type of proliferating cell. INTRODUCTION Since cytogenetic studies of cell p o p u l a t i o n s in acute l y m p h o c y t i c l e u k e m i a (ALL) of c h i l d h o o d s h o w only half the patients to have c h r o m o s o m a l damage [1-9], sister c h r o m a t i d exchange (SCE) provides an a d d i t i o n a l n e w tool for evaluating chromosome damage in ALL. Changes in SCE frequencies and patterns m a y reveal earlier or more subtle changes in the c h r o m o s o m e s than those e v i d e n c e d b y c h r o m o s o m e rearrangement. Such changes might then be of prognostic value. In previous studies from this laboratory we have d e m o n s t r a t e d that c h i l d r e n with ALL have an increased frequency of SCE in p h y t o h e m a g g l u t i n i n (PHA)-stimnlated p e r i p h e r a l blood leukocytes w h e n c o m p a r e d to age-matched control subjects [10]. The increased SCE values are present at diagnosis, before therapy, and rem a i n as long as the c h i l d r e n are on therapy. However, the SCE frequencies observed in stimulated blood leukocytes return to control levels w i t h i n 8 months after therapy has been discontinued. The distribution of SCEs, both inter- and intrachromosomal, in c h i l d r e n w i t h ALL differs from the distribution in control subjects. In the earIier studies from this laboratory we found that, although c h i l d r e n w i t h ALL had l e u k e m i a cells in their p e r i p h e r a l blood at diagnosis, we could not ascertain w h e t h e r the increased SCE occurred in l e u k e m i a cells and/or in residual normal cells. Since l e u k e m i a cells do not r e s p o n d well to PHA [11,12], and the hatFrom the Departments of Medical Genetics and Pediatrics, Indiana University School of Medicine, Indianapolis. Address requests for reprints to Dr. Nyla A. Heerema, Indiana University School of Medicine, Department of Medical Genetics, 1100 W. Michigan, Indianapolis, IN 46223. Received June 8, 1981; accepted November 6, 1981.
323 © Elsevier Science Publishing Co., Inc., 1982 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics6, 323-330 (1982) 0165-4608/82/080323-0852.75
324
Heerema et al. vest and fixation of the cultured peripheral blood cells took place at a time (96 hr) when leukemia cells should no longer be proliferating, we suspected that these responding cells may have been normal circulating lymphocytes. In order to define more precisely the cells with increased SCE, i.e., normal and/ or malignant cells, we examined SCE in bone marrow cells of children with newly diagnosed ALL. Bone marrow was studied, since it was nearly replaced by lymphoblasts. PHA-stimulated as well as unstimulated (spontaneously dividing) marrows were examined to relate the bone marrow studies to the earlier peripheral blood studies. Cell cycle analyses were included to determine if the increase we had seen might be related to a cycle of longer duration in ALL patients.
MATERIALS AND METHODS
The patients studied in this investigation were children with newly diagnosed ALL. None had received any medication. Bone marrow was aspirated for diagnostic purposes from the iliac crest into a heparinized syringe, and a portion of this marrow was used for cytogenetic studies. Two to 0.7 ml of separated lymphocytes (patients 4, 6, 7, 8, controls 1, 3, 4) or whole bone marrow (patients 1, 2, 3, 5, control 2) was cultured in medium consisting of Eagle's minimal essential medium (MEM) with 20% Gibco fetal calf serum, glutamine, nonessential amino acids, and 10 -4 M bromodeoxyuridine (BrdU). Both PHA-stimulated and unstimulated preparations were studied. All cultures were maintained at 37°C in the dark for 72 hr. Three hours before fixation, Velban (0.001 ~.g/ml) was added. After hypotonic treatment with 0.7% sodium citrate, the cells were fixed with 3:1 methanol-acetic acid fixative and dropped onto cold, wet slides. Staining for SCE distribution was accomplished by the method of Korenberg and Freedlender [13]. Twenty well-differentiated second-division metaphases were photographed, and the SCE for each subject was analyzed from projected negatives. Metaphases with fuzzy, ill-defined chromosomes were included in the analysis, as the chromosomes in ALL are notable for their poor morphology [8]. Marrows were obtained from 15 patients with newly diagnosed disease. Of these, 8 had sufficient clearly defined second-division metaphases for analysis after PHA stimulation, but only 4 had adequate spontaneously dividing metaphases in the second division. Control material consisted of bone marrow from one normal adult male, two hematologically normal former ALL patients (one male, 5 years after chemotherapy, and one female, 3 years after chemotherapy), and one 12-year-old female with a localized rhabdomyosarcoma of the eye and hematologically normal marrow. Differential staining of chromosomes that have incorporated BrdU may be used to establish whether the observed metaphases are in the first, M 1 (all darkly stained), second, M 2 (harlequin staining), or third, M 3 (approximately one-fourth darkly and three-fourths lightly stained chromatids) division in vitro. Approximately 100 metaphases per subject were analyzed in this regard. Statistical analyses were performed using the t test for paired observations for two independent samples (based on the log of the SCE values), analysis of variance for repeated measurements, and chi-square tests. RESULTS SCE in Bone Marrow of ALL Patients and Control Subjects
Clinical data at diagnosis for ALL patients in this study are shown in Table 1. One patient, with trisomy 21 Down syndrome, was the only patient not achieving complete remission and is the only patient now deceased.
325
Elevated SCE in Bone Marrow in C h i l d h o o d ALL
Table 1
Patient clinical data
Patient
Sex
Age (yr)
WBC ( × 103/mm3)
Bone marrow blasts (%)
Hemoglobin (g/dl)
Platelets ( × 103/mm3)
Cell surface markers °
1 2 3b 4 5 6 7 8
F F F M M F F M
4 3 10 8 4 2 3 12
3.4 5.2 175.0 18.7 7.0 1.8 19.7 1.0
74 50 99 86 86 80 95 91.5
9.3 5.2 9.7 12.7 8.9 8.7 5.0 11.7
138 37 56 235 272 215 15 42
N N N T N -N N
~N, Null cell; T, T cell. ~l'risomy21, deceased.
The results of the SCE studies and cell cycle analyses are s h o w n in Table 2. The SCE frequencies are calculated on a per c h r o m o s o m e basis because of a n e u p l o i d clones found in ALL, as well as the inclusion of a patient w i t h trisomy 21 and ALL in this study. I n d i v i d u a l s w i t h Down's s y n d r o m e and without l e u k e m i a p r e v i o u s l y have been s h o w n to have normal SCE frequencies [14]. In the stimulated marrows, h y p e r d i p l o i d cells were seen in four of the eight patients; in two, there was extensive aneuploidy. SCE frequencies per c h r o m o s o m e ranged from 0.177 to 0.327 for the patients, with a m e a n of 0.272 SCE per chromosome. The control SCE frequencies were 0.140-0.196, with a m e a n of 0.163 SCE per chromosome. The patient and control means differed significantly (p < 0.001), but there was some heterogeneity. W h e n the patients are c o n s i d e r e d individually, patient 8 d i d not have elevated SCE rates; and the SCE frequency for patient 7, although significantly higher than the control m e a n (p < 0.05), was m u c h lower than that of the other patients and had a value similar to that for one of the control subjects. The significance of these near-normal SCE levels is not clear. No differences in clinical findings at diagnosis were found, and the early response of these patients to t h e r a p y has been as good as that of patients w i t h elevated SCE frequencies. Their progress is being carefully m o n i t o r e d to discern any differences between these two patients and the remaining patients who d e m o n s t r a t e d elevated SCE frequencies. In the four samples with a n e u p l o i d y , there was no difference in SCE frequency in the a n e u p l o i d versus the d i p l o i d cells. A study on the distribution of SCEs in stimulated marrow s h o w e d no evidence of a b i m o d a l distribution (Fig. 1). The m e a n SCE value for unstimulated marrows was 0.189 SCE per c h r o m o s o m e for patients and 0.116 for control subjects. All the SCE frequencies of the i n d i v i d u a l u n s t i m u l a t e d patient marrows (p < 0.05), as well as the m e a n (p < 0.001), were significantly elevated w h e n c o m p a r e d to the control values. In all subjects u n s t i m u l a t e d marrows s h o w e d an SCE frequency lower than that in the PHA-stimulated marrows (p < 0.05). Analysis of variance d e m o n s t r a t e d that the differences in SCE frequency b e t w e e n control and patient values were the same in u n s t i m u l a t e d and PHA-stimulated samples.
Cell Cycle Analysis in ALL and Control Bone Marrows in Vitro The possibility that the observed SCE differences might be related to differences in the cell cycle in control and patient material was c o n s i d e r e d by c o m p a r i s o n of the frequency of cells in first, second, and third division in control subjects and leu-
326 Table 2
H e e r e m a et al.
SCE a n d cell c y c l e i n p a t i e n t s a n d c o n t r o l s a Percentage of cells in
Subject Conffols 1 2 3 4b
Aneuploidy (%)
Cuture conditions
M1
Ms
M3
0 21 42
0 78 46
0 1 12
SCE/chromosome ratio
Unstimulated 0 0 0 .
.
.
.
0 0.134 0.100
. Mean 0.116
Conffols 1 2 3 4
0 0 0 0
Stimulated
Patients 1 2 3 4 5 6 7b 8
0 15.4 0 15.0 0 28.5 . 0
Patients 1 2 3 4 5 6 7 8
0 9 56.3 30.4 14.3 10.0 0 0
30 30 33 32
65 52 33 39
5 18 34 29
0.196 0.165 0.150 0.140 Mean 0.163
0 32 0 33 51 46
0 66 0 64 46 52
0 2 0 3 3 2
0 0.169 0 0.151 0.213 0.249
Unstimulated
.
.
.
. 0
0
0
0 Mean 0.189
1 15 34 33 15 15 4 30
25 67 45 62 55 72 21 45
74 18 21 5 30 13 75 25
0.327 0.319 0.314 0.307 0.272 0.269 0.191 0.177 Mean 0.272
Stimulated
aSee text for statistical significance and P values. bNot tested--insufficient marrow.
k e m i c p a t i e n t s i n b o t h s t i m u l a t e d a n d u n s t i m u l a t e d m a r r o w s . Cell c y c l e a n a l y s i s of s t i m u l a t e d m a r r o w r e v e a l e d h e t e r o g e n e i t y i n b o t h c o n t r o l s u b j e c t s ( p < 0.005) a n d p a t i e n t s (p < 0.005). W h e n p a t i e n t s w e r e c o m p a r e d w i t h c o n t r o l s u b j e c t s , a signifi c a n t d i f f e r e n c e w a s f o u n d ( p < 0.005); c o n t r o l s h a d m o r e c e l l s i n first d i v i s i o n a n d f e w e r i n t h i r d d i v i s i o n , w h e r e a s p a t i e n t s h a d f e w e r c e l l s i n first d i v i s i o n a n d m o r e in t h i r d d i v i s i o n . T h e s e d i f f e r e n c e s , h o w e v e r , w e r e d u e to t h e c o n t r i b u t i o n of t w o p a t i e n t s (1 a n d 7) i n w h i c h 74% a n d 75% of t h e c e l l s w e r e i n t h i r d d i v i s i o n , t h e r e m a i n i n g six p a t i e n t s r e s e m b l i n g c o n t r o l s u b j e c t s i n t h e p e r c e n t a g e of c e l l s i n M 1, M s, a n d M 3. T h e d a t a o n u n s t i m u l a t e d b o n e m a r r o w of b o t h c o n t r o l s u b j e c t s a n d p a t i e n t s s h o w few cells in third division and significant differences a m o n g the control subjects.
327
Elevated SCE in Bone Marrow i n Childhood ALL 4O
30
20
m lo
30--
20
10'
n O
o.
o
o
o
o.~
o
o
o
o
.%o. %
SCEICHROMOSOME
Figure 1 SCE frequencies in individual cells of the patients ([~) and control subjects (,). Both distributions are unimodal; the patients had a much broader distribution and an elevated mean compared to the control subjects. No evidence for two populations of cells--one with control-level SCE and one with elevated SCE--was found in the patients. Dots indicate hyperdiploid cells. SCE frequencies along the entire range were found in hyperdiploid cells.
Comparison of PHA-stimulated versus u n s t i m u l a t e d control marrows showed the stimulated smaples to have significantly more cells in M 3 than the u n s t i m u l a t e d samples (p < 0.005}. In the patients there was also a significant increase in cells in M3 in stimulated as compared to u n s t i m u l a t e d samples, with a concomitant decrease in cells in M 1 (p < 0.005}. These results suggest a faster cell cycle in stimulated versus u n s t i m u l a t e d marrows.
DISCUSSION
The present study shows that an increase in SCE is found i n both stimulated and u n s t i m u l a t e d bone marrow cells of patients with n e w l y diagnosed childhood ALL, confirming the increase found previously in stimulated peripheral blood [10]. The elevated SCE frequency found i n u n s t i m u l a t e d patient marrows shows that the leukemia cells themselves show this p h e n o m e n o n . These studies on ALL are the first to demonstrate an increase in SCE in marrow cells in untreated leukemia. Previous investigations of SCE i n the marrow in other leukemias have s h o w n lower or equivalent frequencies of SCE, but none has demonstrated an increase. In diploid acute myelocytic leukemia (AML} a lower fie-
328
Heerema et al. quency of SCE in bone marrow at diagnosis as c o m p a r e d to that in control marrows or to that in remission was found [15]. A decrease in SCE frequency was demonstrated in marrow cells of untreated chronic myeloblastic l e u k e m i a (CML) patients by Becher et al. [16]. Kakati et al. [17] found no difference in SCE frequency in Ph 1positive CML but did see a significant increase in SCE frequency in two patients in blastic crisis of CML. Knuutila et al. [18] reported no change in SCE frequency in the marrow of patients with CML, AML, refractory i d i o p a t h i c sideroblastic a n e m i a (preleukemia), or malignant histiocytosis as c o m p a r e d to control marrow. However, an increase in SCE was found in marrow cells in megaloblastic a n e m i a [19]. One reason for an increase in SCE m a y be related to the duration of the cell cycle. In stimulated peripheral blood leukocytes of ALL patients, this cycle has been reported to be longer than in control cells, the majority of cells reaching second division only after 96 hr in culture [20]. This m a y result both from a delay and from decreased responses to PHA in m o n o n u c l e a r cells from c h i l d r e n w i t h ALL c o m p a r e d to healthy i n d i v i d u a l s [11,12]. The cell cycle in ALL marrow has also been reported to be slower than in normal cells [21]. In our s t u d y we have not found a d e l a y e d cell cycle in ALL marrow as c o m p a r e d to control marrow. Although the cell cycle in u n s t i m u l a t e d samples was significantly slower than that in stimulated samples, the SCE frequency was greater in the stimulated samples. Thus, we do not believe that the increase in SCE is related to a cell cycle in the ALL cells longer than that in the control cells. ALL at diagnosis and CML in blastic crisis are the only leukemic conditions in w h i c h increases in SCE bone marrow cells have been reported. This suggests a c o m m o n a l i t y in the proliferating cell related to increases in SCE. There have been previous suggestions of differences in SCE levels in different s u b p o p u l a t i o n s of normal h u m a n l y m p h o c y t e s [22-25], and there may be similarities in the types of cells involved in these disorders showing an increase in SCE. Cytomorphological and terminal d e o x y n u c l e o t i d y l transferase studies on CML patients in blastic crisis have shown that in 20% of patients the blasts have l y m p h o i d characteristics [26]. Thus blast cells of CML patients in crisis m a y be indistinguishable from blasts of children with ALL [27]. The l y m p h o c y t e descends from the p l e u r i p o t e n t stem cell w h i c h has been i m p l i c a t e d in CML [28]. These findings suggest that the elevated SCE found in c h i l d h o o d ALL m a y be characteristic of a proliferating l y m p h o c y t e . The increase in SCE we observed in PHA-stimulated cultures as c o m p a r e d to u n s t i m u l a t e d cultures also suggests an effect of PHA; i.e., w h e n different cells are stimulated, different levels of SCE are observed. This agrees with other studies in w h i c h u n s t i m u l a t e d bone marrow had lower SCE frequencies than P H A - s t i m u l a t e d l y m p h o c y t e s [18], and with the observation in our studies on ALL that no difference between stimulated marrow and stimulated peripheral blood [10] was seen. Whatever the cause of the increase in SCE in ALL, it is not exclusive to the leukemia cells, since elevated SCE values are found in stimulated p e r i p h e r a l blood as well as in stimulated and u n s t i m u l a t e d marrow. The difference in SCE frequency in control subjects and patients is the same in both stimulated and s p o n t a n e o u s l y dividing samples. Furthermore, there is no evidence for a b i m o d a l distribution of SCE in stimulated marrow in w h i c h both normal and malignant cells may be present. A n e u p l o i d y (which m a y be indicative of malignant cells) was found in four samples, but there was no difference in SCE frequency per c h r o m o s o m e in the a n e u p l o i d versus the d i p l o i d cells. This is in contrast to acute n o n l y m p h o c y t i c leukemia (ANLL) in w h i c h the a n e u p l o i d leukemic cells were found to have a low SCE frequency (SCEs per cell) as c o m p a r e d to the d i p l o i d cells with w h i c h they coexisted or the d i p l o i d cells found in remission [29]. Since SCE reflects a type of DNA damage [30-32], there may be an increase in DNA damage in ALL that occurs as part of the leukemogenic process. This is re-
Elevated SCE in Bone Marrow i n Childhood ALL
329
flected as an increase in the SCE frequency in the leukemic precursor cells. This increase in SCE in ALL patients is present at diagnosis. Patients i n a preleukemic state need to be studied to determine if the increased SCE occurs prior to the onset of clinical symptoms. However, since the earliest clinical symptoms of ALL are nonspecific, it is very difficult to identify the disease in these children. In the eight cases studied, the time from identification of the first symptoms to diagnosis was 3 days to 4 months. Serial study of patients with conditions that predispose to leukemia may help to elucidate the earliest time of SCE increase. The authors are grateful to Stuart Schwartz for expert technical assistance and to Dr. Pao-Lo Yu for aid in statistical analysis. This is publication No. 81-06 from the Department of Medical Genetics, Indiana University School of Medicine, supported by Grants USPHS T32 GM 07468, Amer. Can. In-46-T, and R01 CA 13809.
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