Dicentric Chromosome 17 in Patients with Leukemia

Joseph R. Testa and Bruce C. Cohen

ABSTRACT: A putative isochromosome of the long arm of chromosome #17 was identified in bone marrow cel ls from each of six patients with leukemia. Adequate s a m p l e was available for

detailed study in four patients, and in each of these four cases examination with multiple staining techniques implied that the rearrangement is a dicentric chromosome. dic(17)(p11.2). An asymmetry of constriction at the two centromeric regions suggested that one centromere was inactive. The data presented here suggest that many of the presumed i(17q) markers observed in leukemia may actually represent dicentric rearrangements.

INTRODUCTION

In recent years, improvements in the quality of banded chromosome preparations from leukemia specimens have resulted in more precise identification of the origin of acquired structural alterations. One well known rearrangement is the isochro- mosome of the long arm of chromosome #17, which is often associated with the transition to the blastic phase of chronic myelogenous leukemia (CML) and has been reported in a number of other malignancies, as well [1-3]. It is generally as- sumed that an i(17q) arises by transverse misdivision of the centromere. Clear-cut evidence for such an event has not been found. There has been one report on a healthy, nonleukemic female in whom a lymphocyte culture showed nonclonal, presumed sister cells containing complementary isochromosomes, i(17p) and i(17q), which suggests that such misdivision can occur [4]. The presence of two centromeric regions in an acquired i(17q) has been reported in a few cases [5-8] and indicates that breakage in this rearrangement may not always occur precisely at the centromere but, instead, in the proximal end of the short arm. The result is a dicentric chromosome with a short intercentromeric region.

Many of the previous studies on patients with i(17q) have relied solely on results obtained with G- or Q-banding alone, which may be insufficient to distinguish two proximate centromeres. In this report we present cytogenetic findings on six cases with putative i(17q) markers studied with multiple staining techniques.

MATERIALS AND METHODS

Six patients with a so-called i(17q] were seen during a 3-year period. Three patients had acute lymphoblastic leukemia (ALL), and three had CML. Cytogenetic analyses were performed on bone marrow aspirates or unstimulated peripheral blood speci- mens at the time of diagnosis in patients with ALL and at blastic crisis in patients

Address requests for reprints to Joseph R. Testa, Ph.D., Section of Cancer Genetics, University of Mary- land Cancer Center, 655 West Baltimore Street, Room 9-031 BRB, Baltimore, MD 21201.

Received August 24, 1985; accepted October 16, 1985.

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© 1986 Elsevier Science Publishing Co., Inc. Cancer Genet Cytogenet 23:47-52 (1986} 52 Vanderbilt Ave., New York, NY 10017 0165-4608/86/$03.50

48 J.R. Testa and B. C. Cohen

with CML. Chromosome preparations were obtained from 24-hour cultures, with or without methotrexate synchronization. Metaphase cells were examined with con- ventional Giemsa, destained, and then reexamined with quinacr ine (Q) banding technique[9]. Some slides were C-banded according to the procedure of Sum- ner[10].

RESULTS

A summary of the karyotypic findings in the six patients is presented in Table 1. A putative i(17q) was identified in each case using Q-banding technique. In patients 1-3 convent ional Giemsa staining of the marker 17 revealed one primary constric- tion and a second, less distinct constriction. Results with C-banding showed two separate positive bands in two of these cases (Fig. 1) and a doubly thick, positive band in the third case (patient 3).

In patient 4, convent ional Giemsa staining of the marker showed a prominent primary constriction and only a slight indenta t ion at a site corresponding to that of the second centromere (Fig. 2a). A small specimen was obtained from this patient, and we were unable to do a C-banding study in this case. However, the available mitoses showed relatively elongated chromosomes, and results with Q-banding technique suggested that the marker was a dicentric chromosome with a small, but discernible amount of moderately fluorescent chromatin between the two centro- meres (Fig. 2b). In patient 5 only a few metaphase cells (6%) contained a presumed i(17q), and no such cell showed adequate C-bands for analysis. In patient 6 we did not have enough sample for C-banding procedures. In addition, the chromosomes in the two latter cases were quite contracted, and it was not possible to determine whether or not the marker was dicentric in these cases based on results with con- ventional Giemsa and Q-banding techniques.

Table 1 Cytogenetic findings in six leukemia patients with putative i(17q)

Patient number Diagnosis ° Specimen b Method ~ Karyotype d

1 CML-BC PB 24 hr

2 CML-BC BM 24 hr

3 CML-BC PB 24 hr MTX

4 ALL BM 24 hr 5 ALL BM 24 hr, 24 hr MTX

6 ALL BM 24 hr, 24 hr MTX

46,XX,t(9;22](q34;q11)(23%)/46,XX,i(17q)*, t(9;22)(38%)/49,XX, + 8, + 19, + 21,i(17q) *, t(9;22)(38%)

46,XX,t(9;22)(q34;q11)(17%)/46,XX,i(17q)*, t(9;22)(83%)

46,XY,t(9;22)(q34;q11)(10%)/46,XY,i(17q)*, t(9;22)(90%)

46,XX,i(17q)*(100%) 46,XY ( 68% )/ 46,XY ,dic( 9 )(p12 )( 26 % )/46,

XY,dic(9),i(17q)(6%) 46,XY(59%)/48,XY, - 1, - 2, + 5, + 16,de1(9)

(p?22),i(17q),t(8;10)(q24;q24), + der(?) t(?;1)(?;p11), + dic(1;2)(p11;p21)(41%)

aALL, acute |ymphoblastic leukemia; CML-BC, chronic myelogenous leukemia in blastic crisis. bBM, bone marrow; PB, peripheral blood. c24 hr refers to cells cultured for 24 hr without synchronization; 24 hr MTX refers to cells cultured for 24 hr and synchronized with methotrexate. ~Available evidence in cases designated by (*) indicates that the rearrangement is a dicentric chromosome, dic(17)(p11.2).

dic(17) in Leukemia 49

Figure 1 C-banded partial metaphase spreads showing two densely stained C-bands (ar- rows) in dicentric chromosomes #17 from (a} patient 1, and (b) patient 2.

DISCUSSION

The occurrence of i(17q) has been associated most commonly with blast ic transfor- mat ion in CML [1-3]. Borgstr6m et al.[8] suggested that loss of part of 17p is a h ighly nonrandom event related to blast ic crisis in CML. In their s tudy of 35 cases of leukemia and pre leukemia wi th unba lanced structural aberrat ions of chromo- some #17, all conta ined loss of the distal part of the short arm. Among these, the p redominan t abnormal i ty was an i(17q).

Evidence avai lable from four of our cases impl ies that the marker 17 is dicentric, and in some mitoses a significant amount of chromosomal mater ial was evident between the two centromeres. We propose that the so-called i(17q) often involves a break at 17p11, most l ikely at subband p11.2. Thus, the abnormal i ty is in terpreted as a dicentr ic chromosome, dic(17)(p11.2). Interest ingly, the adjacent band, 17p12, is known to represent a fragile site [11], and several investigators have suggested that fragile sites and consis tent chromosomal breakpoints in cancer may be related

Figure 2 Chromosome pair #17 from a bone marrow me- taphase cell from patient 4. (a) Conventional Giemsa stain; {b} Q-banding of the same chromosomes. Arrows indicate pre- sumed location of two centromere regions One {? inactive) centromere shows only a slight indentation and exhibits pale staining with Giemsa.

5 0 J.R. Testa and B. C. Cohen

[12-14]. A possible mechanism for the origin of the dic(17) is illustrated in Fig. 3. Such a mechanism of dicentric isochromosome formation would involve an in- trachromosomal rearrangement. It is also possible that the marker 17 could arise by a nonreciprocal translocation between two chromosomes #17, with breakpoints at p11.2 in each chromosome. Because one normal #17 is also present in these cells, the latter mechanism would appear to require a gain of a #17 prior to the translo- cation event. With molecular techniques it should now be possible to determine if the arms of a dic(17) originated from the same maternal of paternal homolog. Some translocations might occur between different homologs, whereas, a dicentric iso- chromosome would always arise from a rearrangement of a single homolog.

Although the relationship to the dic(17) is uncertain, it may be noteworthy that two of our six patients had other dicentrics in addition to the abnormality of chro- mosome #17 (Table 1, patients 5 and 6). Dicentric chromosomes other than the dic(17) appear to be relatively uncommon in leukemia. Patient 5 had a dic(9)(p12), and this abnormality could have originated in a manner similar to that described above for the dic(17).

I

I )

Figure 3 (a) Normal centromere division of chromosome #17; and (b) a possible mecha- nism for the origin of dicentric chromosome #17. In this model, breakage occurs in the proximal short arm of #17, producing a dic(17)(q11.2) and an acentric fragment; the latter is presumably lost. Modified after de la Chapelle[4].

qp -g-

9

a b

dic(17) in Leukemia 5 1

The presence of two centromeres in a single chromosome would seem to com- pl icate segregation of chromosomes at anaphase. It has been proposed that stable dicentr ic chromosomes may conta in one active and one inact ive centromere[15]. Centromere dot (Cd) staining in other s tudies of const i tu t ional dicentr ic chromo- somes has revealed only one active centromere[16] in such rearrangements, sug- gesting that the funct ion of the second centromere is suppressed dur ing cell divi- sion. We a t tempted to perform Cd-banding in our s tudy but were unsuccessful , poss ib ly due to a difference be tween our s tandard hypoton ic swell ing and fixation procedures and those r ecommended by Eiberg[17] for Cd-banding. We used speci- men that had been stored for at least several weeks in methanol /acet ic acid (3:1) fixative, whereas, the method descr ibed by Eiberg involves a very short hypoton ic t reatment (5 min) fol lowed by exposure to 9:1, 5:1, and 3:1 fixative changes and immedia te prepara t ion of slides. Unlike Cd staining or new ant ik inetochore anti- body techniques[18], C-banding does not specif ical ly local ize the centromere itself. However, C-banding does reveal the const i tut ive he terochromat in located at the per icentromeric regions, so that the presence of two deeply s ta ined C-bands in marker 17 is suppor t ive of the in terpreta t ion of a dicentr ic rearrangement. More- over, convent ional Giemsa staining in each of our suspected dicentr ic cases seemed to indicate a difference in the degree of constr ic t ion at the sites of the two centro- meres (Fig. 2a), leading to the conclus ion that one of the two centromeres may be inactive.

Because bone marrow chromosomes are often very contracted and relat ively fuzzy in appearance, it can be difficult to d is t inguish two centromeres in close proximity, especia l ly when Cd- or C-banding has not been performed. There have been previous reports descr ibing the presence of two centromeric regions in an i(17q) [5-8]. Fur thermore, two pat ients wi th acute promyelocyt ic leukemia have been descr ibed in whom an unusual dic(17q-) was ident if ied [19, 20[. The rear- rangement in each of the latter cases showed two C-bands and was thought to be der ived from the 17q-, which prev ious ly had ar isen by a t (15q+ ;17q-). It has been suggested that a higher inc idence of dicentr ic chromosomes might exist in pre- sumed i(17q) cases than has been repor ted prev ious ly wi thout the use of C-banding technique [7]. Our findings wi th C-banding in a small series of pat ients indicate that many, if not all, of the i(17q) markers in leukemia may actual ly represent di- centric chromosomes, and i l lustrate further the impor tance of apply ing C- or Cd- banding or ant ik inetochore ant ibody techniques in s imilar si tuations. Among the three pat ients in our s tudy for whom C-banding was not done, the dicentr ic nature of the rearrangement was apparent in only one case using other s taining techniques.

Supported in part by PHS Grant 1P5OCA 32107 awarded by the National Cancer Institute, DHHS.

Dr. Testa is a Scholar of the Leukemia Society of America, Inc. The authors are grateful to Maureen F. Murtha for expert technical assistance. We also

thank C. David Cash for preparing the photographs and Florence Wade for secretarial assis- tance.

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