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 und n()rma] cells. The m e a l l p e r c e n t a g e aJ 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(t declined slighlly after 72 horn's ill CtlJtill'e lot ALl, pu t Jen 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 st imulation by culture condit ions [8]. However, in ALL, some investigators observed that abnormal clones were most

From the Section of Hematology/Ontology, Department ot Medicine, Llniversity of 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.

93

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94 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 synchronizat ion method or cultured with condit ioned medium 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 addit ional 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 medium (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 9 5

31 PATIENTS WITH AML

1 0 0 %

~ 50%

E

O%

. . . . . . . . . . . _~ . o-'--'--

. . . . . . . . . ..~--~..~ , . . ~ ~ ~ r l

Direct 2 4 4 8 o r

T i m e in C u l t u r e ( h o u r s ) 72 ~

D i a g n o s i s

R e s i d u a l d i s e a s e

R e l a p s e s a m p l e s

Figure I 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 independent 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 individual 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 when 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

96 Y.-S. Li et al.

37 PATIENTS WITH ALL

100%-

"~ 50% /

S .~ / /

o g

O% Direct 24 48

o r

Time in Cu l ture ( h o u r s ) 72 ~

F i g u r e 2 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 subtypes , w h i c h may reflect the smal l n u m b e r of pat ients in each subtype. Five of 8 pa t ien ts w i th M4 l eukemia and all 5 pat ients wi th M5 l eukemia subtype had an increase in abnormal cei ls after cul ture .

Par t icu lar c h r o m o s o m a l abnormal i t i e s may be associa ted wi th an increase in abnor- mal cel ls after cul ture . In AML, 7 of 9 pat ients wi th a greater than 50% increase had a t(8;21)(q22;q22),t{15:17)(q22;q11 - 12), or abnormal i t i es i nvo lv ing 11q23. The remain ing two pat ients had a ~ 4 or t{5:15)((13t:q15). None of these abm)nnal i t i es was seen in 8 pat ients wi th no change or a decrease in abnormal cells. The 8 pat ients 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 ex t remely c o m p l e x karyotype with del{5)(q33q35) and t(9:?) (q34:?).

In 37 pa t ien ts wi th ALL, the percentage of abnormal cel ls increased after cu l ture in 26 pa t ien ts and decreased [9] or l eve led off [2] in 11. The m e a n percentage of abnormal cel ls was found to have increased s ignif icant ly after cu l ture (23% vs. 50%, t - 4.6, p < 0.001) us ing a pa i red t-test. Based on 34 patients , the signif icant d i f ference r e m a i n e d w h e n on ly the 24-hour cul ture was compared wi th the direct m e t h o d (22% vs. 46%, t = 3.7, p < 0.001.). A greater than 50% increase in abnormal cells was obse rved in 9 pa t ien ts and a s ignif icant decrease (> 50%) was noted in on ly two (Fig. 2). The m e a n percen tage of abnormal me taphase cells increased, w i th a peak at 48 hours of cu l tu re and a sl ight dec l ine at 72 hours. When pat ients s tud ied at re lapse and wi th res idua l d isease were exc luded , the percentage of abnormal cel ls increased in 22 pa t ien ts and decreased or leve led off in 9. Because the exc lus ion of pat ients

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 97

T a b l e 1 T h e ave rage 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 cel ls in d i r ec t p r e p a r a t i o n s a n d s h o r t - t e r m c u l t u r e s

Processing

AML" Mean percentage

No. of of abnormal cells Preparations with only samples (+- S.E.) normal cells (%]

Preparations with only abnormal (:ells {'~.,,)

Bone M a r r o w

l)irect 33 37.1 + 6.27.{? 5/33 (15%) j' 2'33 (6%) i' 24 h 114 56.5 + 3.3% 15:114 (13%) 11/114 (10%] 48 h 78 62.6 + 4,1% 4/78 (5%) 18/78 (23%] 72 h 21 67.7 + 6.6% I' 0/21 o. ~, {t) ,.,,) 6,'21 (29%1" t) value < 0.005 0.075 0.(128 Tota l ' Ma r row 120 57.5 ± 3.0% 24,246 (9.8%] 37,246 (15%)

Blood 9 50.4 ~ 13.7%

ALl," Nl(tall [)el'cen lage

No. of {)t almormal (:ells Pr(q)arali()llS with ()nh I)r(![)arali(ms with ()nlv l>ro(:essing samples {+ S.E.I normal (:ells ('{i,) abn()rmal cells ['!;,)

B o n e M a r r o w

Direct 43 27.5 + 4.7% j' 16/43 (37%) I' 0"43 ((,.,(,)1, 24 h 74 44.1 +- 3.7% 9/74 (12%) 3,'74 (4% I 48 h 30 64.7 + 5.6% ~' 3/30 (1()%) ~' 4'30 {13%) ~' 72 h 8 61.0 +- 10.4% 1:8 (13%) 0,8 (()%1*' p value < 0.01 0.007 0.025

Total ' Marrow 86 44.8 + 2.7% 2,(}/155 (19%) 7 155 (4.5%1 Blood 11 59,2 +_ 9.5%

" AML. a(:ute myeloid leukemia: ALL, a(:ute lymphocytic leukemia. *' The p values are (:omparisons betwe(m the highest and the lowest figures in each column. ' Total ind[t;at(is number of [}alien| sanlp[es. S()m(} patients were studied more/hill) on(](!: others wer(~ studied using both bone marrow and peripheral blood.

s t u d i e d at r e l ap s e or w i t h r e s i d u a l d i sease does not a l te r ou r resul t s , t hese p a t i e n t s are i n c l u d e d in the f o l l o w i n g ana lys i s .

In ALL, all 8 p a t i e n t s w i t h over 50 c h r o m o s o m e s in t he i r ce l ls had an i nc r ea se in a b n o r m a l cel ls af ter cu l tu re . For the 11 pa t i en t s w i t h 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 cel ls , t h r ee h a d a b n o r m a l i t i e s i n v o l v i n g 12p11, two e a c h had t r a n s l o c a t i o n s i n v o l v i n g 9q34 or 2 2 q l l { inc lud ing one w i t h a t(9;22), a n d one had an e x t r e m e l y c m n p l e x ka ryo type . T he on ly two pa t i en t s w i th a grea ter t h a n 50% dec rea se in a b n o r n l a l (:ells af ter ( :u l ture bo th ha(l a t r ans loca t io l l i n v o l v i n g 11q13, n a m e l y t(11;?)(q13;?), or d e l ( 1 2 ) ( p l l p 1 3 ) a n d t (11;22)[q13;q11) .

R e p r o d u c i b i l i t y c o u l d not a d e q u a t e l y be i nves t i ga t ed in th i s s tudy . On ly four p a t i e n t s h a d b o t h d i rec t a n d c u l t u r e ana ly se s on two or more ser ia l s amp le s . Inc reases in the p e r c e n t a g e of a b n o r m a l cel ls were seen after cu l t u r e at b o t h s a m p l i n g t i m e s in 3 of 4 pa t i en t s .

Analys i s of Data for Al l P a t i e n t s

Tab le 1 a n d F igure 3 s h o w the m e a n 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 cel ls at d i f f e ren t c u l t u r e t imes . Becaus e succes s fu l cy togene t i c s tud ie s we re p e r f o r m e d on two or more preparations cultured at different lengths of t ime in all patients in this

98 Y.-S. Li et al.

2 0 - ALL

1 0 - -

09 t - O

. I

o..

3 0 - O

..Q

E 2 0 -

Z

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AML

1 o - ~ ~:::: ....... ~ ~ l!!i?!!!iiii~ 1

0 10 20 30 40 50 60 70 80 90 100%

P e r c e n t C h r o n l o s o m a l l y A b n o r m a l Cel ls

Figure 3 A bar graph sho~vit~!-tlm immberof preparations with varying percentages ofchromo- somally abnormal cells in ,,\1,1, HIHt ,\Nil. The, stippled l)ortion of each bar represents r(!lapse saltt pies.

study, the existing chr,mnosomall~ abuormal or normal cells might be observed in one culture but not ()bserved in another from the same sample. The proportion of preparations with oulv m)rmal cells {the chromosomally abnormal cells were undetected) and the pr()t)ortion (if preparations with only abnormal cells (chromosom- ally normal cells were tm(h~tected] is sht)wn in Table 1. The proportion of preparations with only at)normal ceils im:re.sed with culture time, which was consistent with the increasing mean per(:entage of abnorntal cells in AML and ALL. Figure 3 shows the distribution of samples h,, the t~el~ rutage, of ( bromosomallv abnormal cells observed in AML and ALL.

As shown in Table i, {he mean percentage of abnormal cells was lower in the peripheral blood than in the bone marrow in AML. This is consistent with previous reports that in patients known to have an abnormal clone, cytogenetic studies using blood were significantly more, frequently normal than were those using bone marrow 1131. AML samples from !.)nr m.rr(Jw sh(~we(I a significantly higher mean percent- age of at)normal (:ells [57.7%) lhan (lid /\l,I, s,lnH)les (44.8%)(t = 3.07, I) < 0.005). In direct preparations, tewer AML than ALL samples have only normal (:ells (15% vs. 37%,X2 = 4.5, p - : 0.005). As shown in Figure 3. patients with AMLappeared to have more samples containing 80% or more ~:hr(mlosomally at)normal cells than did patients with ALL (43°/6 vs. 16% X z - 17.7, 1) < 0.001).

Processing Leukemia Cells Affects Karyotype c t()

D I S C U S S I O N

Based on data from a large series of patients, we have clearly demunstrated tbal the mean percentage of chromosomally abnormal cells significantly increased wit!~ culture time in both AML and ALL. As a result, for the majority of patients, the likelihood of detecting the abnormal clones also increased. Only two of our 6~1 pat ien ts (both ALL) with both a direct preparation and a cultured sample showed a signiti( aal decrease in abnormal cells after cullure (p - 0.0003 and 0.017, Fisher exact probability test). The percentage of abnormal ,:ells decreased or remained constant after cullurt, in 19 of otlr 68 (28%) patients. Knuutila et al. [31 reported a similar per(:entage ut cytogenetic studies. 6 of 24 (25%), that yielded fewer atmormal (:ells after culture: their patients bad diverse hematologic diseases.

The [)roporfion of chromosomally abnormal (:ells appeared to be <loselv relaled 1(~ their karyotypes, and much less related to the FAB subtypes. Om stud3 does n. t support the previous report [41 that chrom()sonlally abnormal (:ells tl'~m/A,\lXh)l, (N141 subtype do not increase after ( ulture. In our series, 5 of H NI4 pat ients had all ira:re.s. in abnormal (:ells after culture.

Thus. the almormal (:ells from patients with a 1(8:21 l, I[15;17]. and .bn()llnalilit>. involving 11q23 ha(t a very l i g l g r o w t h l)otential whereas the lowest was mHe(] il: two patients with 11q13 translo(:ations. Berger el al. [5, 61 delnunslrah,.d thai llw erythroblasts from patients with a t(8:21 ), t(15:17), and abnormalities involving 11(12:{ were (:hromosomally normal and were dividing only in direct preparations. The erythroblasts from patients with a t(8;21), t(15:17), and abnormalities involving 1 I(t23 were chromosomally normal and were dividing only in direct preparations. The in (:ultured samples in our study. Leukemic (:ells with 5/(lel(Sq). 7/([el(7qL .l (:omplex karyotypes that are asso(:iale(I with nmltil)le cell lineage inv()h em{.d, wou hi be expecte(l to show less pr(q)ortional (:hange. ()ur observal ion that a I,rger i~r~)p()rl ion of AMI. than ALl. saml)les have greater than fill% abnormal karyotypes is ~:{msislenl with other studies. For example, based on the data of the Fourtl~ ,ml Thir~l lnter..a tiomd Workshops on (]hromosomes in Leukemia [13. 141 . the rati()(d ,,\A to AN karyotyt)es was 70% (163 of 232) in AML and only 42% (63 (H 1531 in ALL, ()ur statisti(:al analysis of these data sh()wed them to I)e statisti(:allv significant .t p O.OO5.

The analysis of the complex factors that influence the differential proliferation ot some populations of cells, both leukemic and non-leukemic, in bone marrow samples is beyond the scope of this retrospective study. The goal of a clinical (:ancer (:ytogenet- i ts laboratory is the identification of a clonal chromosomal abnormality. Our results indicate that the likelihood of detecting a clonal chromosome abnormality in leukemi(: samples, both lymphoid and myeloid, increases with culture times up to 48 or 72 hours.

This research was supported by grants from the Department of Energy (DE-FGO2-86ER604081 and the National Institutes of Health (CA 14599, RM) and {CA 42557, JDR]. Dr. Le Beau is , Scholar of the Leukemia Society of America.

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1 0 0 Y.-S. Li et al.

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