Normal Cytogenetic Values for Bone Marrow Based on Studies of Bone Marrow Transplant Donors Daniel G. Kuffel, Cloann G. Schultz, Robert C. Ash, and Gordon W. Dewald
ABSTRACT: For individuals suspected of having hematologic neoplasms, interpretation of the clinical significance of sporadic cells with chromosome breakage, structural anomalies, aneuploidy, or polyploidy is often difficult. To help resolve this problem, we established normal cytogenetic values for bone marrow (BM) by investigating 219 BM transplant (BMT) donors using standard techniques for chromosome analysis. The donors ranged in age from 2 to 58 years and were studied for 7 years. The constitutional karyotype for two individuals was 47,XXY; one was mos4 5 ,X / 46,XX, one was mos46,XX / 4 7 ,XX, + mar, and 215 were normal. Among other statistics, the median and normal ranges (95th percentile) were determined for any kind of chromosome abnormality, autosomal loss, autosomal gain, sex chromosome loss, sex chromosome gain, chromosome breaks or gaps, major structural abnormalities, and polyploidy. The results suggest that random loss of chromosomes is common in cytogenetic preparations of BM, appears to be largely technical and is inversely proportional to chromosome size. Cells with extra chromosomes or with structural abnormalities are rare in normal BM. No specific sporadic structural abnormalities of chromosomes are associated with normal BM. The widely accepted cytogenetic definition for an abnormal clone appears to be valid, with the possible exception of occasional studies involving loss of smaller autosomes. There may be a correlation between loss of the Y chromosome and age of the patient.
INTRODUCTION
In clinical practice, cytogeneticists frequently face difficult decisions about the potential significance of apparent excess breakage, u n u s u a l aneuploidy, or n u m e r o u s polyploid cells in BM specimens. These cytogenetic problems are particularly difficult to resolve w h e n the results border on accepted definitions for an abnormal clone. Because BM is relatively difficult and painful to collect, it may not be surprising that the literature lacks data about normal cytogenetic values for BM. Despite this complication, most laboratory certification agencies require cytogenetic laboratories to establish normal cytogenetic values for use in their clinical practice. Availability of normal cytogenetic values for BM would be helpful in interpretation of the significance of u n u s u a l findings in patients suspected of having clonal neoplas-
From the Cytogenetics Laboratory (D. G. K., C. G. S., G. W. D.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota,and the MilwaukeeCountyMedical Complex (R. C. A.). Milwaukee,Wisconsin. Address reprint requests to: Dr. Gordon W. Dewald, C.ytogenetics Laboratory, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Received November 2, 1990; accepted ]anuary 2, 1991.
39 © 1991 Mayo Foundation
Cancer Genet Cytogenet55:39-48 (1991)
40
D.G. Kuffel et al. tic hematologic disorders. Consequently, we attempted to establish normal cytogenetic values for BM by investigating specimens from 219 BM transplant (BMT) donors.
MATERIALS AND METHODS
Since 1982, we have performed chromosome studies on BM samples from i n d i v i d u a l s being considered as BMT donors and periodically monitored the results as part of our ongoing quality-control program. Our laboratory provides a cytogenetic service for several BMT programs in a d d i t i o n to the one at our own institution. Most of the specimens in this study were collected off site and mailed to us; most were from the Milwaukee County Medical Complex. This study is based on BM specimens processed according to a protocol for mailed-in specimens [1], direct and short-term (24-48 hours) culture method with or without a c t i n o m y c i n D [2], or a direct and short-term (24-48 hours) culture method using e t h i d i u m b r o m i d e and robotic harvesting [3, 4]. Each s p e c i m e n was analyzed using QFQ-banding. Since 1989, we have routinely studied a few cells from each case with distamycin A and 4 ' , 6 - d i a m i d i n o - 2 - p h e n y l i n dole (DA/DAPI). Each s p e c i m e n was examined for clonal c h r o m o s o m e abnormalities and for chromosome p o l y m o r p h i s m s that could be used as markers for engraftment and monitoring remission in the post-BMT period. In each case, we analyzed up to 30 metaphases to define any c h r o m o s o m e a n e u p l o i d y and c h r o m o s o m e breakage. We d e t e r m i n e d the incidence of p o l y p l o i d cells for each case based on up to 100 consecutive metaphases. For purposes of our quality-control program, we defined major structural abnormalities as gross c h r o m o s o m e rearrangements that could potentially survive mitosis, such as c h r o m o s o m e deletions and translocations. Breaks and gaps were defined as m i n o r structural abnormalities. Each chromosome abnormality was described according to the 1985 International System for Cytogenetic Nomenclature (ISCN) [5]. Our definition for an abnormal clone is based on the ISCN criteria and on analysis of up to 30 metaphases for any given subject. An abnormal clone was defined as two or more metaphases with the same h y p e r d i p l o i d karyotype or the same structural abnormality or as three or more metaphases with the same h y p o d i p l o i d karyotype. Eight cytogenetic parameters were d e t e r m i n e d for each individual: the n u m b e r of metaphases with 1) any kind of c h r o m o s o m e abnormality, 2) autosomal loss, 3) autosomal gain, 4) sex chromosome loss, 5) sex chromosome gain, 6) c h r o m o s o m e breaks or gaps, 7) major structural abnormalities, and 8) a p o l y p l o i d karyotype. To normalize the data, the results for each case were expressed in percentage of the cells analyzed. Each variable was tested for bias by sex with W i l c o x o n ' s rank-sum test and by age with S p e a r m a n ' s rank correlation coefficient test. For each cytogenetic parameter, we calculated the median, mean, SD, actual range, and normal range. The "normal range" for each parameter was defined as those numbers less than or equal to the 95th percentile limit. This was calculated for each variable by first ranking the numbers for all the i n d i v i d u a l s in descending order and then establishing the u p p e r 5.0% level. RESULTS
Chromosome studies were done on BM specimens from 219 BMT donors processed in our laboratory from 1982 through 1988. The study i n c l u d e d 110 males and 109 females; their ages ranged from 2 to 58 years (median, 33 years). Four i n d i v i d u a l s had congenital c h r o m o s o m e abnormalities: two were 47,XXY, one was mos45,X/46,XX, and one was mos46,XX/47,XX, + mar (the marker was an abnormal s u p e r n u m e r a r y chromosome of u n k n o w n m a k e u p and was smaller than a c h r o m o s o m e 21). Analysis of the cytogenetic data according to sex and age s h o w e d no apparent
41
N o r m a l C y t o g e n e t i c V a l u e s for B o n e M a r r o w
Table 1
C y t o g e n e t i c r e s u l t s for n o r m a l b o n e m a r r o w Results (%)
Variable Abnormal cells Autosomal loss Autosomal gain Sex chromosome loss F M Sex chromosome gain F M Cells with breaks or gaps Major structural abnormality Polyploidy
No. of subjects
Median
Mean
SD
Actual range
Normal range °
219 219 219 219 109 110 219 109 110 219
10.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
12.93 10.69 0.02 1.03 0.90 1.16 0.02 0.00 0.03 1.09
10.04 8.55 0.34 2.56 2.21 2.87 0.23 0.00 0.32 2.15
0.0-61.1 0.0-50.0 0.0-5.0 0.0-16.7 0.0-10.0 0.0-16.7 0.0-3.3 0.0-0.0 0.0-3.3 0.0-9.7
0.0-30.0 0.0-25.0 0.0-0.0 0.0-5.0 0.0-5.0 0.0-6.0 0.O-0.0 0.0-0.0 0.0-0.0 0.0-5.0
219
0.0
0.16
0.84
0.0-6.3
0.0-0.0
219
0.0
0.62
1.09
0.0-6.0
0.0-3.0
~'95th percentile limit.
s t a t i s t i c a l b i a s e s for a n y of t h e c y t o g e n e t i c v a r i a b l e s . T h u s , w e p o o l e d t h e r e s u l t s for all i n d i v i d u a l s to m a x i m i z e t h e s a m p l e size for s t a t i s t i c a l a n a l y s i s . T h e m e d i a n , m e a n , SD, a c t u a l range, a n d n o r m a l r a n g e for e a c h v a r i a b l e are s u m m a r i z e d i n T a b l e 1. In all, 4,699 m e t a p h a s e s w e r e e x a m i n e d . T h e p r e v a l e n c e of loss or g a i n for e a c h c h r o m o s o m e is s u m m a r i z e d i n F i g u r e 1. W e o b s e r v e d o n l y t w o (0.04%) h y p e r d i p l o i d m e t a p h a s e s : o n e w i t h t r i s o m y 7 a n d t h e o t h e r w i t h XYY. W e o b s e r v e d 514 (10.94%) h y p o d i p l o i d m e t a p h a s e s : o n e m e t a p h a s e h a d 37 c h r o m o s o m e s , t w o h a d 38, t w o h a d 39, t h r e e h a d 40, five h a d 41, 18 h a d 42, 45 h a d 4 3 , 1 0 2 h a d 44, a n d 336 h a d 45. T h e
Figure I donors.
Chromosome aneuploidy in 4,699 metaphases from 219 bone marrow transplant
80 09
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70 60
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50
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40
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Chromosomes gained [ ] Chromosomes lost
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20 10 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 22
Chromosome
X
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42
D.G. Kuffel et al.
6 [] Loss of X in females (17/109 pt) 5
Eo 8
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IB LOS...~So f Y in male.__..~s(19/110 pt)
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0 2 4 21 22 24 25 26 28 29 30 32 33 34 35 36 37 38 39 41 42 43 44 47 48 52 54 58
Age, yr Figure 2 X chromosome loss in 2,239 metaphases from 109 females (17 of 109) and Y chromosome loss in 2,460 metaphases from 110 males (19 of 110), according to age. Each bar represents an individual.
pattern of chromosome loss suggests a correlation between the n u m b e r of cells lacking a particular chromosome and chromosome size. The cells more frequently lacked smaller chromosomes than larger ones. We compared the frequency of sex chromosome loss with the prevalence of autosome loss. To do this, we compensated for the fact that males and females have a different sex chromosome composition (XX and XY) but that each cell should contain two copies of any autosome. In determining the correction factors, we considered that this series of individuals was comprised of approximately 50% (110) males and 50% (109) females. Consequently, we added 25% of the observed frequency of X chromosome loss to the observed value of chromosome loss for the X chromosome. We added 50% of the observed frequency of Y chromosome loss to the observed value of loss for the Y chromosome. The actual and corrected frequencies for both X and Y chromosomes are shown in Figure 1 along with the prevalence of chromosome loss for each of the autosomes. The corrected frequency of X chromosome loss was comparable to chromosomes of similar size, such as chromosomes 7 and 8. The adjusted frequency of Y chromosome loss was comparable to autosomes of similar size, such as chromosomes 19, 20, 21, and 22. For both males and females, we attempted to determine whether any correlation existed between sex chromosome loss and patient age. Among the females, of 2,239 metaphases examined, 18 (0.8%) lacked an X chromosome; these cells occurred among 17 individuals. Of 2,460 metaphases from the males, 28 (1.14%) lacked a Y chromosome; these cells occurred among 19 individuals, Among the males, we also found two (0.08%) metaphases lacking an X chromosome. In addition, two males had three or more cells lacking a Y chromosome. In one other male, four metaphases lacked a Y chromosome. In another male, five metaphases lacked a Y chromosome. Figure 2 shows the age of the 36 individuals and the n u m b e r of cells lacking an X chromosome in females and the n u m b e r lacking a Y chromosome in males. Eleven i n d i v i d u a l s had loss of the same autosome in three or more metaphases
43
Normal Cytogenetic Values for Bone Marrow
Table 2
S u m m a r y of h y p o d i p l o i d y in i n d i v i d u a l s lacking the same autosome in three or more cells
Case
Autosome lost in three or more cells
Cells exhibiting autosoma! loss ~
1 2 3 4 5 6 7 8 9 10 11
7 13 15 18 19 20 21 21 21 22 22
( - 7)(- 7 , - 20)(- 7 , - 12,- 15)(- 21)(- 11,- 17)(- 3, 1 2 , - 1 3 , - 1 7 ) ( - 1 3 , - 2 2 ) ( - 1 1 , 13, 1 5 , - 2 0 ) ( - 6 , - 1 3 , - 1 4 , - 1 7 , - 1 8 , - 1 9 ) ( - 1 9 ) ( - 15)(- 15,- 1 8 ) ( - 6 , - 15)(- 18,- 19) ( - 18)(- 18,- 20)(- 14,- 1 8 , - 2 0 , - 2 1 ) ( - 2 , - 17)(- 1 , - 15,-20) ( - 19)(- 19)(- 19)(- 3)(- 1 , - 4 , - 8 , - 10,- 13,- 14,- 17,-22) ( - 20)(- 20/(- 14,- 16,-20)(- 17,- 18)(- 15,- 22)(-15)(- 13) ( - 2 1 ) ( - 2 1 ) ( - 9 , - 1 2 , - 1 6 , - 2 1 ) ( - 8 , 15)(-15) ( - 21)(-21)(- 18,-21) ( - 2 1 ) ( - 2 0 , - 2 1 ) ( - 4 . - 1 1 , - 2 1 ) ( - 19) ( - 22)(- 22)(- 15,- 22)(- 5)( 6)(- 7)(- 21)(- 11,- 11) ( - 2 2 ) ( - 9 , - 2 2 ) ( - 1 4 , - 2 1 , - 2 2 ) ( - 19,- 20)(- 14)(-20)
Each set of parentheses represents the autosomes lost within a single cell.
(Table 2). With the exception of case 5, the pattern of chromosome loss appeared to be r a n d o m because many of the cells lacked other chromosomes as well. In case 5, although a chromosome 19 was the sole anomaly in three metaphases, two other metaphases in this i n d i v i d u a l lacked different chromosomes. Only eight individuals had any metaphases with major structural abnormalities (Table 3). No i n d i v i d u a l had more than one metaphase with a major structural abnormality, and no two i n d i v i d u a l s had the same structural abnormality. Thus, cells with major structural abnormalities are rare in normal BM and appear to be r a n d o m in origin. Eighty m i n o r abnormalities were found, and they occurred among 71 of the metaphases. The chromosome site of these minor abnormalities appeared to be distributed r a n d o m l y throughout the chromosomes (Fig. 3). DISCUSSION Evaluation of BMT donors offers an opportunity to collect important quality-control data for BM from "normal individuals." Although we do not have formal follow-up information on all individuals in this study, we do know that each one was sufficiently normal to serve as a BMT donor. We believe that these i n d i v i d u a l s are generally representative of a normal population from the central United States.
Table 3
Major structural abnormalities in sporadic cells from eight i n d i v i d u a l s
No. of cells
Type of abnormality
1 1 1 1 1 1 1 1
del(2)(q31) del(15)(q22) del(10)(p11.2p13) * mar t(2;19)(q13;q13.1) del(6)(q13q25) der(16)t(16;?)(?q11;?) del(1)(q11)
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•
Normal Cytogenetic Values for Bone Marrow
45
Four of the i n d i v i d u a l s in our series had constitutional c h r o m o s o m e abnormalities, but this finding did not disqualify them as BMT donors. In each of these cases, the referring p h y s i c i a n was unaware of the u n d e r l y i n g congenital c h r o m o s o m e abnormality until after the cytogenetic studies had been performed. These i n d i v i d u a l s were i n c l u d e d in our study, but because our p u r p o s e was to investigate sporadic chromosome abnormalities we did not consider the constitutional abnormalities in the analysis of our data. The finding of four i n d i v i d u a l s with a constitutional a b n o r m a l i t y in our series of 219 subjects is higher than w o u l d be expected among n e w b o r n populations. This may be a consequence of sampling error. Alternatively, BMT donors are often relatives of BMT recipients and the incidence of congenital c h r o m o s o m e abnormalities may be higher among relatives of patients with neoplastic disorders than it is in normal populations. L a c k of Modern Normal Cytogenetic Values for BM We know of no reports that describe cytogenetic studies of BM from a large series of normal individuals. A few investigations have attempted to establish normal cytogenetic values for BM by studying patients who have undergone BM aspiration p r i m a r i l y for nonmyeloproliferative disorders [6-8]. At least two of these studies i n c l u d e d a few subjects who were normal volunteers [6, 8]. In a d d i t i o n to the early BM investigations, several studies have a t t e m p t e d to establish normal cytogenetic values for peripheral blood and fibroblasts [9-11]. These reports all indicate the presence of some r a n d o m aneuploidy, occasional metaphases with c h r o m o s o m e breakage, and a few p o l y p l o i d cells in different tissues. We are also aware that different tissues and laboratory procedures may be associated with special c h r o m o s o m e abnormalities; e.g., tetraploidy is c o m m o n in amniotic fluid and spontaneous abortuses [12, 13]. Certain sporadic abnormalities, such as 7;14 translocations [14] and inversions of c h r o m o s o m e 14 [15], are c o m m o n in peripheral blood specimens cultured in m e d i u m with certain mitogens. Clones with cells that lack Y chromosomes have been believed to be associated with normal BM [16]. Thus, we a n a l y z e d our data to investigate loss of the X and Y chromosomes and the possibility of specific c h r o m o s o m e abnormalities associated with BM and to establish the normal range for eight cytogenetic parameters. Validity of the Common Definition for an Abnormal Clone The ISCN definition of an abnormal clone is w i d e l y accepted among cytogeneticists; however, it does not specify the number of metaphases or the n u m b e r of s p e c i m e n s on w h i c h its definition is based. That most laboratories routinely study 2 0 - 3 0 metaphases for any particular specimen suggests that most cytogeneticists t h i n k in terms of these numbers w h e n they use the definition. We analyzed up to 30 metaphases for each i n d i v i d u a l in our study (average, 21 metaphases). We did not study BM from any i n d i v i d u a l in our series more than once. Our data generally support the definition for an abnormal clone involving h y p o d i p loidy. Considering the great n u m b e r of h y p o d i p l o i d metaphases in our series, only one case involving h y p o d i p l o i d y of an autosome w o u l d have presented a diagnostic dilemma. Case 5 had five abnormal metaphases: three had m o n o s o m y 19 as the sole anomaly, and two were lacking other c h r o m o s o m e s (Table 2). These results probably do not reflect a hematologic disorder in this i n d i v i d u a l because the subject was clinically normal and had no apparent hematologic disease. In addition, m o n o s o m y 19 alone is rarely associated with hematologic disorders [17]. This case may demonstrate that the definition of a h y p o d i p l o i d clone is imperfect. Our results for autosomal h y p e r d i p l o i d y and for structural abnormalities indicate
46
D.G. Kuffel et al. that these kinds of chromosome anomalies are relatively rare. None of our subjects w o u l d have represented a diagnostic d i l e m m a with respect to either of these two cytogenetic parameters. For cases in which the cytogenetic results are questionable, further c h r o m o s o m e studies, perhaps later, w o u l d be of interest to determine whether an abnormal clone is emerging. These additional studies might be especially important if a classic chromosome abnormality is involved, such as m o n o s o m y 7 or trisomy 8. The usefulness of this type of protocol in clinical practice has been described in other publications
[18]. "Normal Range" for Cytogenetic Parameters We d e c i d e d that the simplest approach to establish the normal range for our data was to use the 95% values for each data set. Thus, in routine practice, any results higher than the 95th percentile could be considered outside the " n o r m a l limits" for our laboratory and should raise suspicions of an abnormal clone. This outcome itself w o u l d not automatically make the result abnormal, however. Other factors, such as whether the chromosome abnormality is associated with hematologic disorders, should also be considered. The m e d i a n loss of one or more autosomes was 10% of the metaphases, and the normal range was 0 - 2 5 % (Table 1). Thus, if more than 25% of metaphases show random loss, one of the following may be suspected: a technical problem, a premalignant process [19], unstable centromeres, m y e l o s u p p r e s s i v e treatment, exposure to occupational clastogens, or other biologic factors that may cause c h r o m o s o m e loss. Our results suggest that the frequency of loss for each chromosome may be related to chromosome size because small chromosomes were lost more frequently than large chromosomes (Fig. 1). A similar pattern of chromosome loss has been described for other tissues [10, 11, 20]. Because the pattern of chromosome loss a p p e a r e d to be r a n d o m except for an association with chromosome size, and because the incidence was considerably greater than h y p e r d i p l o i d y and structural abnormalities, we believe that chromosome loss in these i n d i v i d u a l s resulted primarily from technical circumstances. The results of our study indicate that the gain of one or more autosomes in any metaphase is rare and that the normal limit is 0%. Therefore, it w o u l d be reasonable to suspect an abnormal clone involving h y p e r d i p l o i d y when the m i n i m a l criterion for the definition of an abnormal clone is satisfied. It may even be reasonable to suspect an abnormal clone w h e n only one metaphase with trisomy is observed, especially if the extra chromosome is one that has been associated with hematologic disorders. In our experience, it is not u n u s u a l to see p o l y p l o i d metaphases in specimens from i n d i v i d u a l s suspected of having hematologic disorders. We studied i n d i v i d u a l s in w h o m the proportion of metaphases that were tetraploid exceeded 50%, and no structural c h r o m o s o m e abnormalities suggested further karyotypic abnormalities. The results of our study indicate that the normal range for p o l y p l o i d y is 0 - 3 . 0 % of metaphases, although one i n d i v i d u a l in our series had 6% p o l y p l o i d metaphases. Thus, observation of more than 3.0% p o l y p l o i d metaphases could raise concern about the possibility of a neoplastic p o l y p l o i d clone or some unusual proliferation of normal cells tetraploid in nature. Major structural abnormalities were also rare in our study; the normal range for our subjects was 0%. Thus, the w i d e l y accepted definition for an abnormal clone involving metaphases with structural abnormalities appears to be adequate to detect clonal processes. Our data suggest that observation of one or more cells with a major
Normal Cytogenetic Values for Bone Marrow
47
structural abnormality would be u n u s u a l for a normal population. Thus, any cells with a major abnormality might raise concerns about the presence of an abnormal clone or about the patient having been exposed to toxic substances or having a chromosome breakage syndrome. Similar abnormalities may occasionally be due to technical artifact. The normal range for minor structural abnormalities among our i n d i v i d u a l s was 0-5.0%. Thus, more than 5% of metaphases with chromosome breaks or gaps may be evidence of a technical problem, a chromosome breakage syndrome, or exposure to substances that cause chromosome breakage. We looked for evidence of special "hot spots" on chromosomes for minor chromosome breakage but were not impressed with any specific breakpoints.
X and Y C h r o m o s o m e Loss
The subject of Y chromosome loss has been extensively discussed in the literature, but results remain conflicting. Some investigators suggest that loss of a Y chromosome in BM cells may be part of the normal aging process [16]. Other investigators suggest that loss of the Y chromosome may sometimes be part of an u n d e r l y i n g clonal neoplastic process [21, 22]. We suspect that both viewpoints may be correct; thus, in some patients loss of Y chromosomes may not be clinically significant, but in others it may be part of a neoplastic process. Several reports on lymphocytes have suggested a significant increase in loss of X chromosomes in females and Y chromosomes in males after age 55 years [9, 23]. Studies on BM from m e n with no primary hematologic disease have shown 45,X, - Y cell lines in those 60 years or older [6, 7]. The m e d i a n age of i n d i v i d u a l s in our series was only 33 years. The relatively young age of our subjects may have biased our attempts to investigate properly the associations of chromosome loss and age. Among the females, our study indicated no apparent correlation between X chromosome loss and increasing age. Among the males, in two individuals, one aged 30 and one aged 44 years, two cells lacked a Y chromosome. Two other males, one aged 43 and one aged 58 years, lacked a Y chromosome in four or more cells, and they were among the oldest males in our series. Although our data set is small, the results may suggest a correlation between the age of males and the possibility of finding a clone of cells lacking a Y chromosome. If so, our data suggest that a true clone of cells missing a Y chromosome can occur in BM of some normal individuals. We were surprised that among the males only two metaphases were lacking an X chromosome. This incidence is considerably less than loss of X chromosomes in the females (18 of 2,239 metaphases) and autosomes of comparable size, even after correction is made for the difference in sex chromosome composition. We do not know whether this finding is due to small sample size, sampling error, influencing biologic factors, or some combination of these problems. Among the 2,460 metaphases from males in our series, 28 lacked a Y chromosome as compared with only two cells lacking an X chromosome, a 14-fold difference. The difference in the size of the X and Y chromosomes may account for m u c h of this discrepancy, but it does not account for the total difference. The greater loss of Y chromosomes than X chromosomes among the males may suggest that factors other than random loss are involved. In males, cells lacking a Y chromosome may be able to survive but cells lacking an X may not.
We thank Drs. Robert B. Jenkins and Syed M. Jalal for their helpful suggestions.
48
D . G . Kuffel et al.
REFERENCES 1. Dewald G, Allen JE, Strutzenberg DK, Pierre RV (1982)'. A cytogenetic method for mailedin bone marrow specimens for the study of hematologic disorders. Lab Med 13:225-229. 2. Dewald GW, Broderick DJ, Tom WW, Hagstrom JE, Pierre RV (1985): The efficacy of direct, 24-hour culture, and mitotic synchronization methods for cytogenetic analysis of bone marrow in neoplastic hematologic disorders. Cancer Genet Cytogenet 18:1-10. 3. Spurbeck JL, Carlson RO, Allen JE, Dewald GW (1988): Culturing and robotic harvesting oI bone marrow, lymph nodes, peripheral blood, fibroblasts, and solid tumors with in situ techniques. Cancer Genet Cytogenet 32:59-66. 4. Tomiyasu T, Testa JR (1988): Application of ethidium bromide for high-resolution banding analysis of chromosomes from h u m a n malignant cells. Stain Technol 63:83-91. 5. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP (eds.); (1985): ICSN 1985. Also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985. 6. O'Riordan ML, Berry EW, Tough IM (1970): Chromosome studies on bone marrow from a male control population. Br J Haematol 19:83-90. 7. Walker LMS (1971): The chromosomes of bone-marrow ceils of haematologicalty normal men and women. Br J Haematol 21:455-461. 8. Pierre RV, Hoagland HC (1972): Age-associated aneuploidy: Loss of Y chromosome from h u m a n bone marrow cells with aging. Cancer 30:889-894. 9. Galloway SM, Buckton KE (1978): Aneuploidy and aging: Chromosome studies on a random sample of the population using G-banding. Cytogenet Cell Genet 20:78-95. 10. Wenger SL, Golden WL, Dennis SP, Steele MW (1984): Are the occasional aneuploid cells in peripheral blood cultures significant? Am J Med Genet 19:715-719. 11. Wenger SL (1989): Nonmodal chromosome gain and loss in h u m a n fibroblast cultures. Cytogenet Cell Genet 52:201. 12. Milunsky A (1979): The prenatal diagnosis of chromosomal disorders. In: Genetic Disorders and the Fetus, A Milunsky, ed. Plenum Press, New York, pp. 93-156. 13. Tharapel AT. Tharapel SA, Bannerman RM (1985): Recurrent pregnancy losses and parental chromosome abnormalities: a review. Br J Obstet Gynecol 92:899-914. 14. Dewald GW, Noonan KJ, Spurbeck JL, Johnson DD (1986): T-lymphocytes with 7;14 translocations: Frequency of occurrence, breakpoints, and clinical and biological significance. Am J Hum Genet 38:520-532. 15. Aurias A, Dutrillaux B (1986): Acquired inversions in h u m a n leucocytes. Ann Genet 29:203-206. 16. Pierre RV, Hoagland HC (1971): 45,X cell lines in adult men: Loss of Y chromosome, a normal aging phenomenon? Mayo Clin Proc 46:52-55. 17. Mitelman F (19881: Catalog of Chromosome Aberrations in Cancer, 3rd ed. Alan R. Liss, New York. 18. McConnell TS, Duncan MH, Foucar K, and the Southwestern Oncology Group Leukemia Cytogenetics Subcommittee: Do random [non-clonal] chromosome abnormalities in bone marrow predict a clone to come? Cancer Genet Cytogenet (in press). 19. Johnson GA, Dewald GW, Strand WR, Winkelmann RK (1985): Chromosome studies in 17 patients with the S~zary syndrome. Cancer 55:2426-2433. 20. Neurath P, DeRemer K, Bell B, Jarvik L, Kato T (1970): Chromosome loss compared with chromosome size, age and sex of subjects. Nature 225:281-282. 21. Sakurai M. Sandberg AA (1976): The chromosomes and causation of h u m a n cancer and leukemia: XVIII. The missing Y in acute myeloblastic leukemia (AML) and phi-positive chronic myelocytic leukemia (CML). Cancer 38:762-769. 22. Abe S, Golomb HM, Rowley JD, Mitelman F, Sandberg AA (1980): Chromosomes and causation of h u m a n cancer and leukemia: XXXV. The missing Y in acute non-lymphocytic leukemia (ANLL). Cancer 45:84-90. 23. Sandberg AA, Cohen MM, Rimm AA, Levin ML (1967): Aneuploidy and age in a population survey. Am J Hum Genet 19:633-643.
ABSTRACT: For individuals suspected of having hematologic neoplasms, interpretation of the clinical significance of sporadic cells with chromosome breakage, structural anomalies, aneuploidy, or polyploidy is often difficult. To help resolve this problem, we established normal cytogenetic values for bone marrow (BM) by investigating 219 BM transplant (BMT) donors using standard techniques for chromosome analysis. The donors ranged in age from 2 to 58 years and were studied for 7 years. The constitutional karyotype for two individuals was 47,XXY; one was mos4 5 ,X / 46,XX, one was mos46,XX / 4 7 ,XX, + mar, and 215 were normal. Among other statistics, the median and normal ranges (95th percentile) were determined for any kind of chromosome abnormality, autosomal loss, autosomal gain, sex chromosome loss, sex chromosome gain, chromosome breaks or gaps, major structural abnormalities, and polyploidy. The results suggest that random loss of chromosomes is common in cytogenetic preparations of BM, appears to be largely technical and is inversely proportional to chromosome size. Cells with extra chromosomes or with structural abnormalities are rare in normal BM. No specific sporadic structural abnormalities of chromosomes are associated with normal BM. The widely accepted cytogenetic definition for an abnormal clone appears to be valid, with the possible exception of occasional studies involving loss of smaller autosomes. There may be a correlation between loss of the Y chromosome and age of the patient.
INTRODUCTION
In clinical practice, cytogeneticists frequently face difficult decisions about the potential significance of apparent excess breakage, u n u s u a l aneuploidy, or n u m e r o u s polyploid cells in BM specimens. These cytogenetic problems are particularly difficult to resolve w h e n the results border on accepted definitions for an abnormal clone. Because BM is relatively difficult and painful to collect, it may not be surprising that the literature lacks data about normal cytogenetic values for BM. Despite this complication, most laboratory certification agencies require cytogenetic laboratories to establish normal cytogenetic values for use in their clinical practice. Availability of normal cytogenetic values for BM would be helpful in interpretation of the significance of u n u s u a l findings in patients suspected of having clonal neoplas-
From the Cytogenetics Laboratory (D. G. K., C. G. S., G. W. D.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota,and the MilwaukeeCountyMedical Complex (R. C. A.). Milwaukee,Wisconsin. Address reprint requests to: Dr. Gordon W. Dewald, C.ytogenetics Laboratory, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Received November 2, 1990; accepted ]anuary 2, 1991.
39 © 1991 Mayo Foundation
Cancer Genet Cytogenet55:39-48 (1991)
40
D.G. Kuffel et al. tic hematologic disorders. Consequently, we attempted to establish normal cytogenetic values for BM by investigating specimens from 219 BM transplant (BMT) donors.
MATERIALS AND METHODS
Since 1982, we have performed chromosome studies on BM samples from i n d i v i d u a l s being considered as BMT donors and periodically monitored the results as part of our ongoing quality-control program. Our laboratory provides a cytogenetic service for several BMT programs in a d d i t i o n to the one at our own institution. Most of the specimens in this study were collected off site and mailed to us; most were from the Milwaukee County Medical Complex. This study is based on BM specimens processed according to a protocol for mailed-in specimens [1], direct and short-term (24-48 hours) culture method with or without a c t i n o m y c i n D [2], or a direct and short-term (24-48 hours) culture method using e t h i d i u m b r o m i d e and robotic harvesting [3, 4]. Each s p e c i m e n was analyzed using QFQ-banding. Since 1989, we have routinely studied a few cells from each case with distamycin A and 4 ' , 6 - d i a m i d i n o - 2 - p h e n y l i n dole (DA/DAPI). Each s p e c i m e n was examined for clonal c h r o m o s o m e abnormalities and for chromosome p o l y m o r p h i s m s that could be used as markers for engraftment and monitoring remission in the post-BMT period. In each case, we analyzed up to 30 metaphases to define any c h r o m o s o m e a n e u p l o i d y and c h r o m o s o m e breakage. We d e t e r m i n e d the incidence of p o l y p l o i d cells for each case based on up to 100 consecutive metaphases. For purposes of our quality-control program, we defined major structural abnormalities as gross c h r o m o s o m e rearrangements that could potentially survive mitosis, such as c h r o m o s o m e deletions and translocations. Breaks and gaps were defined as m i n o r structural abnormalities. Each chromosome abnormality was described according to the 1985 International System for Cytogenetic Nomenclature (ISCN) [5]. Our definition for an abnormal clone is based on the ISCN criteria and on analysis of up to 30 metaphases for any given subject. An abnormal clone was defined as two or more metaphases with the same h y p e r d i p l o i d karyotype or the same structural abnormality or as three or more metaphases with the same h y p o d i p l o i d karyotype. Eight cytogenetic parameters were d e t e r m i n e d for each individual: the n u m b e r of metaphases with 1) any kind of c h r o m o s o m e abnormality, 2) autosomal loss, 3) autosomal gain, 4) sex chromosome loss, 5) sex chromosome gain, 6) c h r o m o s o m e breaks or gaps, 7) major structural abnormalities, and 8) a p o l y p l o i d karyotype. To normalize the data, the results for each case were expressed in percentage of the cells analyzed. Each variable was tested for bias by sex with W i l c o x o n ' s rank-sum test and by age with S p e a r m a n ' s rank correlation coefficient test. For each cytogenetic parameter, we calculated the median, mean, SD, actual range, and normal range. The "normal range" for each parameter was defined as those numbers less than or equal to the 95th percentile limit. This was calculated for each variable by first ranking the numbers for all the i n d i v i d u a l s in descending order and then establishing the u p p e r 5.0% level. RESULTS
Chromosome studies were done on BM specimens from 219 BMT donors processed in our laboratory from 1982 through 1988. The study i n c l u d e d 110 males and 109 females; their ages ranged from 2 to 58 years (median, 33 years). Four i n d i v i d u a l s had congenital c h r o m o s o m e abnormalities: two were 47,XXY, one was mos45,X/46,XX, and one was mos46,XX/47,XX, + mar (the marker was an abnormal s u p e r n u m e r a r y chromosome of u n k n o w n m a k e u p and was smaller than a c h r o m o s o m e 21). Analysis of the cytogenetic data according to sex and age s h o w e d no apparent
41
N o r m a l C y t o g e n e t i c V a l u e s for B o n e M a r r o w
Table 1
C y t o g e n e t i c r e s u l t s for n o r m a l b o n e m a r r o w Results (%)
Variable Abnormal cells Autosomal loss Autosomal gain Sex chromosome loss F M Sex chromosome gain F M Cells with breaks or gaps Major structural abnormality Polyploidy
No. of subjects
Median
Mean
SD
Actual range
Normal range °
219 219 219 219 109 110 219 109 110 219
10.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
12.93 10.69 0.02 1.03 0.90 1.16 0.02 0.00 0.03 1.09
10.04 8.55 0.34 2.56 2.21 2.87 0.23 0.00 0.32 2.15
0.0-61.1 0.0-50.0 0.0-5.0 0.0-16.7 0.0-10.0 0.0-16.7 0.0-3.3 0.0-0.0 0.0-3.3 0.0-9.7
0.0-30.0 0.0-25.0 0.0-0.0 0.0-5.0 0.0-5.0 0.0-6.0 0.O-0.0 0.0-0.0 0.0-0.0 0.0-5.0
219
0.0
0.16
0.84
0.0-6.3
0.0-0.0
219
0.0
0.62
1.09
0.0-6.0
0.0-3.0
~'95th percentile limit.
s t a t i s t i c a l b i a s e s for a n y of t h e c y t o g e n e t i c v a r i a b l e s . T h u s , w e p o o l e d t h e r e s u l t s for all i n d i v i d u a l s to m a x i m i z e t h e s a m p l e size for s t a t i s t i c a l a n a l y s i s . T h e m e d i a n , m e a n , SD, a c t u a l range, a n d n o r m a l r a n g e for e a c h v a r i a b l e are s u m m a r i z e d i n T a b l e 1. In all, 4,699 m e t a p h a s e s w e r e e x a m i n e d . T h e p r e v a l e n c e of loss or g a i n for e a c h c h r o m o s o m e is s u m m a r i z e d i n F i g u r e 1. W e o b s e r v e d o n l y t w o (0.04%) h y p e r d i p l o i d m e t a p h a s e s : o n e w i t h t r i s o m y 7 a n d t h e o t h e r w i t h XYY. W e o b s e r v e d 514 (10.94%) h y p o d i p l o i d m e t a p h a s e s : o n e m e t a p h a s e h a d 37 c h r o m o s o m e s , t w o h a d 38, t w o h a d 39, t h r e e h a d 40, five h a d 41, 18 h a d 42, 45 h a d 4 3 , 1 0 2 h a d 44, a n d 336 h a d 45. T h e
Figure I donors.
Chromosome aneuploidy in 4,699 metaphases from 219 bone marrow transplant
80 09
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40
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Chromosomes gained [ ] Chromosomes lost
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20 10 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 22
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42
D.G. Kuffel et al.
6 [] Loss of X in females (17/109 pt) 5
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IB LOS...~So f Y in male.__..~s(19/110 pt)
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0 2 4 21 22 24 25 26 28 29 30 32 33 34 35 36 37 38 39 41 42 43 44 47 48 52 54 58
Age, yr Figure 2 X chromosome loss in 2,239 metaphases from 109 females (17 of 109) and Y chromosome loss in 2,460 metaphases from 110 males (19 of 110), according to age. Each bar represents an individual.
pattern of chromosome loss suggests a correlation between the n u m b e r of cells lacking a particular chromosome and chromosome size. The cells more frequently lacked smaller chromosomes than larger ones. We compared the frequency of sex chromosome loss with the prevalence of autosome loss. To do this, we compensated for the fact that males and females have a different sex chromosome composition (XX and XY) but that each cell should contain two copies of any autosome. In determining the correction factors, we considered that this series of individuals was comprised of approximately 50% (110) males and 50% (109) females. Consequently, we added 25% of the observed frequency of X chromosome loss to the observed value of chromosome loss for the X chromosome. We added 50% of the observed frequency of Y chromosome loss to the observed value of loss for the Y chromosome. The actual and corrected frequencies for both X and Y chromosomes are shown in Figure 1 along with the prevalence of chromosome loss for each of the autosomes. The corrected frequency of X chromosome loss was comparable to chromosomes of similar size, such as chromosomes 7 and 8. The adjusted frequency of Y chromosome loss was comparable to autosomes of similar size, such as chromosomes 19, 20, 21, and 22. For both males and females, we attempted to determine whether any correlation existed between sex chromosome loss and patient age. Among the females, of 2,239 metaphases examined, 18 (0.8%) lacked an X chromosome; these cells occurred among 17 individuals. Of 2,460 metaphases from the males, 28 (1.14%) lacked a Y chromosome; these cells occurred among 19 individuals, Among the males, we also found two (0.08%) metaphases lacking an X chromosome. In addition, two males had three or more cells lacking a Y chromosome. In one other male, four metaphases lacked a Y chromosome. In another male, five metaphases lacked a Y chromosome. Figure 2 shows the age of the 36 individuals and the n u m b e r of cells lacking an X chromosome in females and the n u m b e r lacking a Y chromosome in males. Eleven i n d i v i d u a l s had loss of the same autosome in three or more metaphases
43
Normal Cytogenetic Values for Bone Marrow
Table 2
S u m m a r y of h y p o d i p l o i d y in i n d i v i d u a l s lacking the same autosome in three or more cells
Case
Autosome lost in three or more cells
Cells exhibiting autosoma! loss ~
1 2 3 4 5 6 7 8 9 10 11
7 13 15 18 19 20 21 21 21 22 22
( - 7)(- 7 , - 20)(- 7 , - 12,- 15)(- 21)(- 11,- 17)(- 3, 1 2 , - 1 3 , - 1 7 ) ( - 1 3 , - 2 2 ) ( - 1 1 , 13, 1 5 , - 2 0 ) ( - 6 , - 1 3 , - 1 4 , - 1 7 , - 1 8 , - 1 9 ) ( - 1 9 ) ( - 15)(- 15,- 1 8 ) ( - 6 , - 15)(- 18,- 19) ( - 18)(- 18,- 20)(- 14,- 1 8 , - 2 0 , - 2 1 ) ( - 2 , - 17)(- 1 , - 15,-20) ( - 19)(- 19)(- 19)(- 3)(- 1 , - 4 , - 8 , - 10,- 13,- 14,- 17,-22) ( - 20)(- 20/(- 14,- 16,-20)(- 17,- 18)(- 15,- 22)(-15)(- 13) ( - 2 1 ) ( - 2 1 ) ( - 9 , - 1 2 , - 1 6 , - 2 1 ) ( - 8 , 15)(-15) ( - 21)(-21)(- 18,-21) ( - 2 1 ) ( - 2 0 , - 2 1 ) ( - 4 . - 1 1 , - 2 1 ) ( - 19) ( - 22)(- 22)(- 15,- 22)(- 5)( 6)(- 7)(- 21)(- 11,- 11) ( - 2 2 ) ( - 9 , - 2 2 ) ( - 1 4 , - 2 1 , - 2 2 ) ( - 19,- 20)(- 14)(-20)
Each set of parentheses represents the autosomes lost within a single cell.
(Table 2). With the exception of case 5, the pattern of chromosome loss appeared to be r a n d o m because many of the cells lacked other chromosomes as well. In case 5, although a chromosome 19 was the sole anomaly in three metaphases, two other metaphases in this i n d i v i d u a l lacked different chromosomes. Only eight individuals had any metaphases with major structural abnormalities (Table 3). No i n d i v i d u a l had more than one metaphase with a major structural abnormality, and no two i n d i v i d u a l s had the same structural abnormality. Thus, cells with major structural abnormalities are rare in normal BM and appear to be r a n d o m in origin. Eighty m i n o r abnormalities were found, and they occurred among 71 of the metaphases. The chromosome site of these minor abnormalities appeared to be distributed r a n d o m l y throughout the chromosomes (Fig. 3). DISCUSSION Evaluation of BMT donors offers an opportunity to collect important quality-control data for BM from "normal individuals." Although we do not have formal follow-up information on all individuals in this study, we do know that each one was sufficiently normal to serve as a BMT donor. We believe that these i n d i v i d u a l s are generally representative of a normal population from the central United States.
Table 3
Major structural abnormalities in sporadic cells from eight i n d i v i d u a l s
No. of cells
Type of abnormality
1 1 1 1 1 1 1 1
del(2)(q31) del(15)(q22) del(10)(p11.2p13) * mar t(2;19)(q13;q13.1) del(6)(q13q25) der(16)t(16;?)(?q11;?) del(1)(q11)
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Normal Cytogenetic Values for Bone Marrow
45
Four of the i n d i v i d u a l s in our series had constitutional c h r o m o s o m e abnormalities, but this finding did not disqualify them as BMT donors. In each of these cases, the referring p h y s i c i a n was unaware of the u n d e r l y i n g congenital c h r o m o s o m e abnormality until after the cytogenetic studies had been performed. These i n d i v i d u a l s were i n c l u d e d in our study, but because our p u r p o s e was to investigate sporadic chromosome abnormalities we did not consider the constitutional abnormalities in the analysis of our data. The finding of four i n d i v i d u a l s with a constitutional a b n o r m a l i t y in our series of 219 subjects is higher than w o u l d be expected among n e w b o r n populations. This may be a consequence of sampling error. Alternatively, BMT donors are often relatives of BMT recipients and the incidence of congenital c h r o m o s o m e abnormalities may be higher among relatives of patients with neoplastic disorders than it is in normal populations. L a c k of Modern Normal Cytogenetic Values for BM We know of no reports that describe cytogenetic studies of BM from a large series of normal individuals. A few investigations have attempted to establish normal cytogenetic values for BM by studying patients who have undergone BM aspiration p r i m a r i l y for nonmyeloproliferative disorders [6-8]. At least two of these studies i n c l u d e d a few subjects who were normal volunteers [6, 8]. In a d d i t i o n to the early BM investigations, several studies have a t t e m p t e d to establish normal cytogenetic values for peripheral blood and fibroblasts [9-11]. These reports all indicate the presence of some r a n d o m aneuploidy, occasional metaphases with c h r o m o s o m e breakage, and a few p o l y p l o i d cells in different tissues. We are also aware that different tissues and laboratory procedures may be associated with special c h r o m o s o m e abnormalities; e.g., tetraploidy is c o m m o n in amniotic fluid and spontaneous abortuses [12, 13]. Certain sporadic abnormalities, such as 7;14 translocations [14] and inversions of c h r o m o s o m e 14 [15], are c o m m o n in peripheral blood specimens cultured in m e d i u m with certain mitogens. Clones with cells that lack Y chromosomes have been believed to be associated with normal BM [16]. Thus, we a n a l y z e d our data to investigate loss of the X and Y chromosomes and the possibility of specific c h r o m o s o m e abnormalities associated with BM and to establish the normal range for eight cytogenetic parameters. Validity of the Common Definition for an Abnormal Clone The ISCN definition of an abnormal clone is w i d e l y accepted among cytogeneticists; however, it does not specify the number of metaphases or the n u m b e r of s p e c i m e n s on w h i c h its definition is based. That most laboratories routinely study 2 0 - 3 0 metaphases for any particular specimen suggests that most cytogeneticists t h i n k in terms of these numbers w h e n they use the definition. We analyzed up to 30 metaphases for each i n d i v i d u a l in our study (average, 21 metaphases). We did not study BM from any i n d i v i d u a l in our series more than once. Our data generally support the definition for an abnormal clone involving h y p o d i p loidy. Considering the great n u m b e r of h y p o d i p l o i d metaphases in our series, only one case involving h y p o d i p l o i d y of an autosome w o u l d have presented a diagnostic dilemma. Case 5 had five abnormal metaphases: three had m o n o s o m y 19 as the sole anomaly, and two were lacking other c h r o m o s o m e s (Table 2). These results probably do not reflect a hematologic disorder in this i n d i v i d u a l because the subject was clinically normal and had no apparent hematologic disease. In addition, m o n o s o m y 19 alone is rarely associated with hematologic disorders [17]. This case may demonstrate that the definition of a h y p o d i p l o i d clone is imperfect. Our results for autosomal h y p e r d i p l o i d y and for structural abnormalities indicate
46
D.G. Kuffel et al. that these kinds of chromosome anomalies are relatively rare. None of our subjects w o u l d have represented a diagnostic d i l e m m a with respect to either of these two cytogenetic parameters. For cases in which the cytogenetic results are questionable, further c h r o m o s o m e studies, perhaps later, w o u l d be of interest to determine whether an abnormal clone is emerging. These additional studies might be especially important if a classic chromosome abnormality is involved, such as m o n o s o m y 7 or trisomy 8. The usefulness of this type of protocol in clinical practice has been described in other publications
[18]. "Normal Range" for Cytogenetic Parameters We d e c i d e d that the simplest approach to establish the normal range for our data was to use the 95% values for each data set. Thus, in routine practice, any results higher than the 95th percentile could be considered outside the " n o r m a l limits" for our laboratory and should raise suspicions of an abnormal clone. This outcome itself w o u l d not automatically make the result abnormal, however. Other factors, such as whether the chromosome abnormality is associated with hematologic disorders, should also be considered. The m e d i a n loss of one or more autosomes was 10% of the metaphases, and the normal range was 0 - 2 5 % (Table 1). Thus, if more than 25% of metaphases show random loss, one of the following may be suspected: a technical problem, a premalignant process [19], unstable centromeres, m y e l o s u p p r e s s i v e treatment, exposure to occupational clastogens, or other biologic factors that may cause c h r o m o s o m e loss. Our results suggest that the frequency of loss for each chromosome may be related to chromosome size because small chromosomes were lost more frequently than large chromosomes (Fig. 1). A similar pattern of chromosome loss has been described for other tissues [10, 11, 20]. Because the pattern of chromosome loss a p p e a r e d to be r a n d o m except for an association with chromosome size, and because the incidence was considerably greater than h y p e r d i p l o i d y and structural abnormalities, we believe that chromosome loss in these i n d i v i d u a l s resulted primarily from technical circumstances. The results of our study indicate that the gain of one or more autosomes in any metaphase is rare and that the normal limit is 0%. Therefore, it w o u l d be reasonable to suspect an abnormal clone involving h y p e r d i p l o i d y when the m i n i m a l criterion for the definition of an abnormal clone is satisfied. It may even be reasonable to suspect an abnormal clone w h e n only one metaphase with trisomy is observed, especially if the extra chromosome is one that has been associated with hematologic disorders. In our experience, it is not u n u s u a l to see p o l y p l o i d metaphases in specimens from i n d i v i d u a l s suspected of having hematologic disorders. We studied i n d i v i d u a l s in w h o m the proportion of metaphases that were tetraploid exceeded 50%, and no structural c h r o m o s o m e abnormalities suggested further karyotypic abnormalities. The results of our study indicate that the normal range for p o l y p l o i d y is 0 - 3 . 0 % of metaphases, although one i n d i v i d u a l in our series had 6% p o l y p l o i d metaphases. Thus, observation of more than 3.0% p o l y p l o i d metaphases could raise concern about the possibility of a neoplastic p o l y p l o i d clone or some unusual proliferation of normal cells tetraploid in nature. Major structural abnormalities were also rare in our study; the normal range for our subjects was 0%. Thus, the w i d e l y accepted definition for an abnormal clone involving metaphases with structural abnormalities appears to be adequate to detect clonal processes. Our data suggest that observation of one or more cells with a major
Normal Cytogenetic Values for Bone Marrow
47
structural abnormality would be u n u s u a l for a normal population. Thus, any cells with a major abnormality might raise concerns about the presence of an abnormal clone or about the patient having been exposed to toxic substances or having a chromosome breakage syndrome. Similar abnormalities may occasionally be due to technical artifact. The normal range for minor structural abnormalities among our i n d i v i d u a l s was 0-5.0%. Thus, more than 5% of metaphases with chromosome breaks or gaps may be evidence of a technical problem, a chromosome breakage syndrome, or exposure to substances that cause chromosome breakage. We looked for evidence of special "hot spots" on chromosomes for minor chromosome breakage but were not impressed with any specific breakpoints.
X and Y C h r o m o s o m e Loss
The subject of Y chromosome loss has been extensively discussed in the literature, but results remain conflicting. Some investigators suggest that loss of a Y chromosome in BM cells may be part of the normal aging process [16]. Other investigators suggest that loss of the Y chromosome may sometimes be part of an u n d e r l y i n g clonal neoplastic process [21, 22]. We suspect that both viewpoints may be correct; thus, in some patients loss of Y chromosomes may not be clinically significant, but in others it may be part of a neoplastic process. Several reports on lymphocytes have suggested a significant increase in loss of X chromosomes in females and Y chromosomes in males after age 55 years [9, 23]. Studies on BM from m e n with no primary hematologic disease have shown 45,X, - Y cell lines in those 60 years or older [6, 7]. The m e d i a n age of i n d i v i d u a l s in our series was only 33 years. The relatively young age of our subjects may have biased our attempts to investigate properly the associations of chromosome loss and age. Among the females, our study indicated no apparent correlation between X chromosome loss and increasing age. Among the males, in two individuals, one aged 30 and one aged 44 years, two cells lacked a Y chromosome. Two other males, one aged 43 and one aged 58 years, lacked a Y chromosome in four or more cells, and they were among the oldest males in our series. Although our data set is small, the results may suggest a correlation between the age of males and the possibility of finding a clone of cells lacking a Y chromosome. If so, our data suggest that a true clone of cells missing a Y chromosome can occur in BM of some normal individuals. We were surprised that among the males only two metaphases were lacking an X chromosome. This incidence is considerably less than loss of X chromosomes in the females (18 of 2,239 metaphases) and autosomes of comparable size, even after correction is made for the difference in sex chromosome composition. We do not know whether this finding is due to small sample size, sampling error, influencing biologic factors, or some combination of these problems. Among the 2,460 metaphases from males in our series, 28 lacked a Y chromosome as compared with only two cells lacking an X chromosome, a 14-fold difference. The difference in the size of the X and Y chromosomes may account for m u c h of this discrepancy, but it does not account for the total difference. The greater loss of Y chromosomes than X chromosomes among the males may suggest that factors other than random loss are involved. In males, cells lacking a Y chromosome may be able to survive but cells lacking an X may not.
We thank Drs. Robert B. Jenkins and Syed M. Jalal for their helpful suggestions.
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REFERENCES 1. Dewald G, Allen JE, Strutzenberg DK, Pierre RV (1982)'. A cytogenetic method for mailedin bone marrow specimens for the study of hematologic disorders. Lab Med 13:225-229. 2. Dewald GW, Broderick DJ, Tom WW, Hagstrom JE, Pierre RV (1985): The efficacy of direct, 24-hour culture, and mitotic synchronization methods for cytogenetic analysis of bone marrow in neoplastic hematologic disorders. Cancer Genet Cytogenet 18:1-10. 3. Spurbeck JL, Carlson RO, Allen JE, Dewald GW (1988): Culturing and robotic harvesting oI bone marrow, lymph nodes, peripheral blood, fibroblasts, and solid tumors with in situ techniques. Cancer Genet Cytogenet 32:59-66. 4. Tomiyasu T, Testa JR (1988): Application of ethidium bromide for high-resolution banding analysis of chromosomes from h u m a n malignant cells. Stain Technol 63:83-91. 5. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP (eds.); (1985): ICSN 1985. Also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985. 6. O'Riordan ML, Berry EW, Tough IM (1970): Chromosome studies on bone marrow from a male control population. Br J Haematol 19:83-90. 7. Walker LMS (1971): The chromosomes of bone-marrow ceils of haematologicalty normal men and women. Br J Haematol 21:455-461. 8. Pierre RV, Hoagland HC (1972): Age-associated aneuploidy: Loss of Y chromosome from h u m a n bone marrow cells with aging. Cancer 30:889-894. 9. Galloway SM, Buckton KE (1978): Aneuploidy and aging: Chromosome studies on a random sample of the population using G-banding. Cytogenet Cell Genet 20:78-95. 10. Wenger SL, Golden WL, Dennis SP, Steele MW (1984): Are the occasional aneuploid cells in peripheral blood cultures significant? Am J Med Genet 19:715-719. 11. Wenger SL (1989): Nonmodal chromosome gain and loss in h u m a n fibroblast cultures. Cytogenet Cell Genet 52:201. 12. Milunsky A (1979): The prenatal diagnosis of chromosomal disorders. In: Genetic Disorders and the Fetus, A Milunsky, ed. Plenum Press, New York, pp. 93-156. 13. Tharapel AT. Tharapel SA, Bannerman RM (1985): Recurrent pregnancy losses and parental chromosome abnormalities: a review. Br J Obstet Gynecol 92:899-914. 14. Dewald GW, Noonan KJ, Spurbeck JL, Johnson DD (1986): T-lymphocytes with 7;14 translocations: Frequency of occurrence, breakpoints, and clinical and biological significance. Am J Hum Genet 38:520-532. 15. Aurias A, Dutrillaux B (1986): Acquired inversions in h u m a n leucocytes. Ann Genet 29:203-206. 16. Pierre RV, Hoagland HC (1971): 45,X cell lines in adult men: Loss of Y chromosome, a normal aging phenomenon? Mayo Clin Proc 46:52-55. 17. Mitelman F (19881: Catalog of Chromosome Aberrations in Cancer, 3rd ed. Alan R. Liss, New York. 18. McConnell TS, Duncan MH, Foucar K, and the Southwestern Oncology Group Leukemia Cytogenetics Subcommittee: Do random [non-clonal] chromosome abnormalities in bone marrow predict a clone to come? Cancer Genet Cytogenet (in press). 19. Johnson GA, Dewald GW, Strand WR, Winkelmann RK (1985): Chromosome studies in 17 patients with the S~zary syndrome. Cancer 55:2426-2433. 20. Neurath P, DeRemer K, Bell B, Jarvik L, Kato T (1970): Chromosome loss compared with chromosome size, age and sex of subjects. Nature 225:281-282. 21. Sakurai M. Sandberg AA (1976): The chromosomes and causation of h u m a n cancer and leukemia: XVIII. The missing Y in acute myeloblastic leukemia (AML) and phi-positive chronic myelocytic leukemia (CML). Cancer 38:762-769. 22. Abe S, Golomb HM, Rowley JD, Mitelman F, Sandberg AA (1980): Chromosomes and causation of h u m a n cancer and leukemia: XXXV. The missing Y in acute non-lymphocytic leukemia (ANLL). Cancer 45:84-90. 23. Sandberg AA, Cohen MM, Rimm AA, Levin ML (1967): Aneuploidy and age in a population survey. Am J Hum Genet 19:633-643.
