The Proportion of Abnormal Karyotypes in Acute Leukemia Samples Related to Method of Preparation You-Sheng Li, Michelle M. Le Beau, Rosemarie Mick, and Janet D. Rowley

ABSTRACT: Bone tnarrow ond peripheral blood were studied trom 20,0 patients with acille leukemia [lilt) rrith acute ms'eloid leuke, mia IAML). 91 with acute 15"lnptn)blastic leakemJa IALIJ] who had salnple, s cultured (or varying limes and who had (I mixture ol cbronlosomallv atmormal u n d n()rma] cells. The m e a l l p e r c e n t a g e a J abnornla] m e / a p h a s e cells incFeased !.l Jttl C(llhlre lime. The peak wus reache, d at 4B hatlrs (1(1(tdeclined slighlly after 72 horn's ill CtlJtill'e lot ALl, p u t J e n t s . The aleall percentage ol" ubaorlnal cells Jacreascd up to 72 horn's Ja (:ill(tire' lor AMI, i)atients. In 68 patients (31 AML and 37 ALL), cytogenetic data were available from samples processed with bath direct preparations and culture methods. The percentage of abnormal cells increased after culture in 49 patients (23 AML and 26 ALL), while it decreased or remained at the same level in 19 patients. For AML patients, the mean percentage of abnormal cells was significantly different between direct (38%) and cultured preparations (63%), (p < 0.001). Seven of 9 patients with AML who showed a greater than 50% increase in abnormal cells after culture had either a t(8;21), t(15;17), orabnormalities involving 11q23. The two patients who showed a significant decrease in abnormal cells both had a translocation involving i lq13. Compared with ALL, more AML patients showed greater than 80% abnormal bone marrow metaphase (:ells at diagnosis oi" at relapse.

INTRODUCTION Cytogenetic studies have become an essential part of research in leukemia and of the clinical management of patients who have malignant hematologic diseases. Whether short term in vitro culture increases the likelihood of detecting a chromosomally abnormal clone of cells has been a matter for debate. Before the 1980s, a generally held view was that in vitro culture might lead to overgrowth of normal cells. With improved culture techniques, many investigators found the contrary to be true [1-4]. Chromosomally normal erythroblasts that were not involved in the leukemia process in some patients frequently stop dividing within a few hours after culture and this can lead to a higher proportion of abnormal cells derived from other cell lineages in cultured preparations [5-7]. Chromosomally abnormal cells may also reenter active proliferation more quickly than normal cells after stimulation by culture conditions [8]. However, in ALL, some investigators observed that abnormal clones were most From the Section of Hematology/Ontology,Department ot Medicine, Llniversityof Chicago, Chicago. Illinois. Address reprint requests to: Dr. Janet D. Rowley, Department of Medicine, Box 420, 5841 S. Maryland Avenue, Chicago, Illinois 60637. Received June 12, 1990; accepted July 17, 1990.

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Y.-S. Li et al.

often detected in direct preparations [9, 10]. More recently, Dewald et al. [11] found no difference in karyotypes, success rate, or metaphase quality between direct and 24-hour culture methods in a double-blind prospective study on a large series of marrow aspirates from patients with various hematologic disorders. Clearly, more extensive studies are required to resolve this issue. MATERIALS AND METHODS Patients Cytogenetic evaluation of 220 patient samples obtained from a total of 200 patients with acute leukemia (91 with ALL, 109 with AML) provided the data for this study. Patients were selected because they had bone marrow or peripheral blood samples that were processed using at least two different methods, e.g., direct preparations, 24-, 48-, or 72-hour cultures, harvested from at least two different times, and had a mixture of chromosomally normal and abnormal cells. These represented all the bone marrow or peripheral blood samples from patients with acute leukemia received by this laboratory from 1980 to 1988 that met the above criteria. Samples studied using a cell synchronization method or cultured with conditioned m e d i u m were excluded. Of the 95 ALL patient samples evaluated for cytogenetic studies, 75 were obtained at diagnosis, 19 at relapse, and one was obtained during residual disease, i.e., after chemotherapy but before a complete remission. Four patients were studied on two occasions. Of the 125 AML patient samples, 87 were obtained at diagnosis. 23 at relapse, and 15 obtained during residual disease. Thirteen patients were studied twice and one additional patient was studied four times. Because only 26 cytogenetic studies were performed using peripheral blood (6 yielded fewer than 5 photo-analyzed cells) and 14 of them were performed using bone marrow (:ells as well. this paper refers only to bone marrow studies, except in the section that discusses the difference between peripheral blood and bone marrow samples. Cytogenetic Studies Samples were usually processed immediately after aspiration or venipuncture, but a delay in processing of up to a few hours might have occurred if the sample was obtained from an outside hospital. Cells were cultured in 10 ml m e d i u m (96% RPMI 1640, 10% fetal bovine serum, 10 mM HEPES, 100 units/ml penicillin, 100 mcg/ml streptomycin, pH adjusted to 7.2 7.3 with 7.5% sodium bicarbonate) in 25 cm z flasks at 37°C in a humidified 5% CO~ 95% air atmosphere. The cell concentration was 10"/ml. For direct preparations, the cells were incubated for I hour prior to processing; cultured samples were incubated for 24, 48. or 72 hours. C((lcemid (0.05 mcg/ml) was added 10 45 minutes before metaphase cell preparation. Cytogenetic analyses were performed with quinacrine fluorescence and trypsin-Giemsa bandingtechniques. The criteria proposed by Rowley and Potter were used for the identification of abnormal clones [12]. For each preparation, at least 10 and usually 20 or more ceils were photographed and analyzed in detail. Only photo-analyzed cells were included in this evaluation. When different culture times were considered, all preparations in which fewer than five cells were photo-analyzed were excluded. Statistics Separate statistical analyses were conducted for ALL and AML patients. Comparison of mean percent of abnormal cells between direct and culture methods was performed in 68 patients with data from both methods, by a paired t-test. Comparison of mean

Processing Leukemia Cells Affects Karyotype

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Relationship between the percentage of almormal metaphase cells and time of harvest in 31 patients with AML. Thirty-two samples were studied with both a direct preparation and at least one culture time. Lines on the (:hart inarked with an asterisk were cultured for 72 honrs.

percentage of abnormal cells in bone marrow between direct preparations and various cultured samples was evaluated by a t-test for i n d ep en d en t groups. Comparison of proportion of patients with 80% or greater abnormal cells between ALL and AML, comparison of percentage of preparations with only abnormal cells or with only normal cells at different culture times, and comparison of percentage of abnormal cells in i n d i v i d u a l patients at different culture times were all done by the Chi-square test. All tests were two-sided, with significance set at 0.05.

RESULTS

Patients with Both Direct and Cultured Samples To compare the proportion of chromosomally abnormal cells in direct preparations with cultured samples, data were collected from 68 patients who were studied using both methods. In 31 patients with AML, the percentage of chromosomally abnormal cells increased after culture in 23 patients, and it decreased [6] or remained constant [2] in 8 patients (Fig. 1). The mean percentage of abnormal cells was found to have increased after culture (38% vs. 63%, t - 4.9, p < 0.001). Based on 26 patients, the difference was still significant w h e n only the 24-hour culture was compared to the direct method (37% vs. 63%, t - 4.5, p < 0.001). The proportion of abnormal cells after culture decreased less than 20% (0 17%) in all 6 patients and was not statistically significant, whereas the increase was over 50% in 9 patients (Fig. 1). When patients studied at relapse or with residual disease were excluded, the percentage of abnormal cells increased after culture in 20 patients and decreased or leveled off in 4. No apparent difference was seen in the proportion of abnormal karyotypes in different

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37 PATIENTS WITH ALL

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Relationship between the percentage of abnormal metaphase cells and time of harvest in 37 patients with ALL. Thirty-seven samples were studied with both a direct preparation and at least one culture time. I,ines on the chart marked with an asterisk were cultured for 72 hours.

FAB s u b t y p e s , w h i c h m a y reflect the small n u m b e r of patients in each subtype. Five of 8 p a t i e n t s w i t h M4 l e u k e m i a and all 5 patients w i t h M5 l e u k e m i a s u b t y p e had an i n c r e a s e in a b n o r m a l ceils after culture. P a r t i c u l a r c h r o m o s o m a l a b n o r m a l i t i e s m a y be associated w i t h an increase in abnormal cells after culture. In AML, 7 of 9 patients w i t h a greater than 50% increase had a t(8;21)(q22;q22),t{15:17)(q22;q11 - 12), or a b n o r m a l i t i e s i n v o l v i n g 11q23. The r e m a i n i n g two patients had a ~ 4 or t{5:15)((13t:q15). N o n e of these a b m ) n n a l i t i e s was seen in 8 patients with no c h a n g e or a d e c r e a s e in a b n o r m a l cells. The 8 patients had a 18. + 8 , del[14)[q22q32),t[3:21)lq26;q22),t(14:?)[q32:?),inv(16)[p13q22), d e l ( 3 q ) / + 1 3 , or an e x t r e m e l y c o m p l e x karyotype with del{5)(q33q35) and t(9:?) (q34:?). In 37 p a t i e n t s w i t h ALL, the percentage of a b n o r m a l cells i n c r e a s e d after c u l t u r e in 26 p a t i e n t s and d e c r e a s e d [9] or l e v e l e d off [2] in 11. The m e a n p e r c e n t a g e of a b n o r m a l cells was f o u n d to h a v e i n c r e a s e d significantly after c u l t u r e (23% vs. 50%, t - 4.6, p < 0.001) u s i n g a paired t-test. Based on 34 patients, the significant difference r e m a i n e d w h e n o n l y the 24-hour culture was c o m p a r e d w i t h the direct m e t h o d (22% vs. 46%, t = 3.7, p < 0.001.). A greater than 50% increase in a b n o r m a l cells was o b s e r v e d in 9 p a t i e n t s and a significant d e c r e a s e (> 50%) was noted in o n l y two (Fig. 2). T h e m e a n p e r c e n t a g e of a b n o r m a l m e t a p h a s e cells increased, w i t h a peak at 48 h o u r s of c u l t u r e and a slight d e c l i n e at 72 hours. W h e n patients s t u d i e d at relapse and w i t h r e s i d u a l disease w e r e e x c l u d e d , the percentage of a b n o r m a l cells i n c r e a s e d in 22 p a t i e n t s and d e c r e a s e d or l e v e l e d off in 9. Because the e x c l u s i o n of patients

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P r o c e s s i n g L e u k e m i a Cells Affects K a r y o t y p e

T a b l e 1 T h e a v e r a g e p e r c e n t a g e of c h r o m o s o m a l l y a b n o r m a l cells in d i r e c t preparations and short-term cultures

Processing

No. of samples

AML" Mean percentage of abnormal cells Preparations with only (+- S.E.) normal cells (%]

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37.1 56.5 62.6 67.7

The proportion of abnormal karyotypes in acute leukemia samples related to method of preparation.

Bone marrow and peripheral blood were studied from 200 patients with acute leukemia [109 with acute myeloid leukemia (AML), 91 with acute lymphoblasti...
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