Cytogenetic Peculiarities in Chronic Myelogenous Leukemia Frauke Hild and Christa Fonatsch

ABSTRACT: Cytogenetic investigations were performed in 185 patients with chronic myelogenous leukemia (CML) at all stages of the disease; 166 patients were Ph positive--159 (95.8%) of these showing the standard Ph translocation, and 7 (4.2%) variant translocations--17 patients were Ph negative. In 2 patients the cytogenetic analysis was unsuccessful. Additional aberrations were found in 40 (24.1%) of the Ph-positive patients. Nine (52.9%) of the Ph-negative patients showed chromosome anomalies. Besides the well known nonrandom abnormalities ( - 7, + 8, i(17q), + 19. + Ph ) we found a high frequency of clones with rare or not yet described structural rearrangements--in 14 cases (34.2%) of the Ph-positive patients and in 2 cases (20%) of the Phnegative patients with other chromosome abnormalities. The clinical significance of these findings is discussed.

INTRODUCTION The Philadelphia (Ph) translocation represents the characteristic cytogenetic abnormality in chronic myelogeneous leukemia (CML) subdividing the disease into Ph chromosome positive or Ph chromosome negative forms [1-3]. Five to ten percent of CML patients are found to be Ph negative [4-8]. In a distinct majority of the Phpositive cases (92%-95%), the Ph is due to the standard translocation (9;22)(q34;q11), but in 5% to 8% a so-called variant translocation occurs, involving additional chromosomes in the translocation [9-16]. Additional chromosome abnormalities are generally considered to be of poor prognostic value, at the most signifying or accompanying the blastic crisis (BC). They are detected in more than 80% of the patients being in the BC. However, in a small n u m b e r of patients, additional aberrations have been detected early in the chronic phase (CP), and their prognostic significance still remains unclear [17-25]. We present cytogenetic data of 185 CML patients studied in our laboratory. Particular emphasis is put on the cytogenetic characteristics of 47 patients who showed additional and/or secondary aberrations or variant Ph translocations. MATERIALS AND METHODS

From 1983 until 1988, 330 samples of bone marrow and/or peripheral blood from 185 patients with CML (62 females and 123 males) were provided by several hematological clinics for chromosome analysis. In 120 patients, including 14 in BC, a single cytogeFrom the Institut for Humanenetik,MedizinischeUniversit~tzu Lfibeck,RatzeburgerAllee 160, D-2400 Lfibeck 1, Federal Republicof Germany. Address reprint requests to: Dr. Christa Fonatsch, Institut fiJr Humangenetik, Medizinische Universit~t zu L~beck, Ratzeburger Allee 160, D-2400 LfJbeck 1; Telex: 026492 ul; Telefax: 500.3016. Received October 19, 1989; accepted November 14, 1989.

197 © 1990 ElsevierSciencePublishingCo., Inc. 655 Avenueof the Americas,New York, NY 10010

Cancer Genet Cytogenet47:197 217 (1990) 0165-4608/90/$3.50

198

F. Hild and C. Fonatsch

netic analysis was carried out. In 65 patients, 2 to 7 cytogenetic analyses were performed at different stages of the disease. Cytogenetic studies were performed on bone marrow cells a n d / o r peripheral blood cells directly after half an hour of incubation in a Colcemid-containing hypotonic solution, and after short-term cultivation for 24h, 48h, and 72h using different culture m e d i a without mitogens, as described by Stollmann et al. [26]. The constitutional karyotype of the patients was determined from peripheral blood lymphocytes cultured for 72h in the presence of phytohemagglutinin (PHA). Mitoses were routinely analysed using either the GAG-banding technique described by Fonatsch et al. [27] or the fluorescence banding technique (QFQ) of Caspersson et al. [28]. RESULTS Among the 185 cases investigated, no metaphases were found in two the remaining 183 patients, 166 (90.7%) were Ph positive presenting Ph translocation in 159 cases (95.8%) and variant translocations in 7 Forty (24.1%) of the Ph-positive cases showed additional aberrations. patients no Ph c h r o m o s o m e was detected.

cases. Out of the standard cases (4.2%). In 17 (9.3%)

Variant Translocations (Table 1) Cytogenetic and clinical data of the 7 patients with variant Ph translocations are listed in Table 1. Cytogenetic and molecular genetic data, respectively, of patients H. L. and T. M. (cases 2 and 7) have been previously reported in detail. A n involvement of c h r o m o s o m e 9 in the t(22;22) (case 7) was demonstrated by in situ-hybridization [29, 30]. A further variant translocation without a cytogenetically detectable involvement of c h r o m o s o m e 9 was found in patient B. M. (case 1). Complex translocations occurred in patient R. H-P. (case 3), showing two further clones with additional aberrations besides the variant translocation, and in patients G. K., H. P., and M. H. (cases 4-6).

Ph Positive Patients with Additional Aberrations Clonal aberrations in a d d i t i o n to the Ph chromosome were found in forty cases, as s u m m a r i z e d in Tables 2A-C. In one case (patient H. B., case 47), a constitutional triple X was detected. The remaining patients had a normal constitutive karyotype.

Additional aberrations found in the blast crisis (Table 2A): A d d i t i o n a l aberrations in BC were detected in 14 out of 17 patients who were first analysed in the BC, as listed in Table 2A. The cytogenetic data of patient M. S. (case 9) have recently been p u b l i s h e d [29]. Patient D. R. (case 17) showed a duplication of the long arm of c h r o m o s o m e 17,dup(17)(q21--)q25). Patient P. R. (case 20) showed different structural changes: t(1;3)(q23 or 25;q29), del(6)(q12), del(11)(q12), i(17q), and 1 7 q ÷ .

Additional aberrations f o u n d in CP progressing to BC (Table 2B ): Eight patients w h o were studied at least twice showed the clinical characteristics of the chronic phase of CML at the time of the first cytogenetic investigation. Later on they showed progression of the disease a c c o m p a n i e d by karyotypic evolution (Table 2B). Patient D. L. (case 22), first observed in 1986, exhibited the standard Ph translocation. One year later, in the beginning of the acceleration, his bone marrow gained a c h r o m o s o m e 8, and 4 months after that, he presented a third clone with a deletion of the short arm of c h r o m o s o m e 7, del(7)(p13). Patient W. I. (case 23) showed a clone with m o n o s o m y of the short arms of

B.M.

H.L.

R. H-P. G.K. H.P.

1

2

3 4 5

T.M.

M. H.

Patient

02/26/87 03/17/87 09/30/87 02/17/88 03/03/88 01/21/87 02/18/86 05/07/86 12/02/86 06/10/87

08/21/86 05/25/87 11/15/84

Culture date

P M

M F M

F

M

Sex

48 49 34 35 35 45 51 51 51 52

28 29 48

Age

List of variant translocations

Case no.

Table 1

BC CP CP CP AP AP CP CP CP CP

CP CP CP

Status of disease

Busulfan Busulfan Busulfan IFN~2b IFN~2b Busulfan Busulfan Busulfan Busulfan Busulfan

Busulfan Busulfan

Therapy t(2;22)(q37;q11) t(2;22)(q37;q11) t(2;9;22)(p15 or 13;q34;q11) t(7;9;22)(q22;q34;q11) t(9;11;22)(q34;q13;q11) t(9;11;22)(q34;q13;q11) t(9;11;22)(q34;q13;q11) t(9;11;122)(q34;q13;q11) t(9;12;22)(q34;q11/13;q11/ t(22;22)(q11;q13/ t(22;22)(q11;q13) t(22;22)(q11;q13) t(22;22)(q11;q13)

Variant translocations

- Y/t(10;16)(q26;q12),i(17q), ÷ P h

Additional aberrations

N.H. K.W.

K.L.

T.H.

B.H.

J.H.

H.F. D.R. B.E. S.J.

P. R,

10 11

12

13

14

15

16 17 18 19

20

R. H-P.

M.S.

9

3

L.E.

Patient

8

Case no.

Table 2A

02/26/87

09/16/86 11/17/87 09/25/86 11/12/87 02/17/88 10/15/86

01/22/87

08/14/84

M

M M M M M F

M

M

F

F

07/13/87

10/15/87

M F

06/05/86 01/19/87

M

M

11/27/87

04/09/86

M

Sex

09/17/87

Culture date

48

80 48 40 45 46 48

44

41

60

70

? 51

51

53

53

Age

BC

BC AP BC AP AP AP

BC

BC

BC

BC

BC BC

BC extramedullary BC extramedullary BC extramedullary

Status of disease

Busulfan

Busulfan Busulfan Busulfan Busulfan iFNa2b Busulfan

Busulfan

Busulfan

Hydroxycarbamid

Busulfan Bus.,Vin., Lit. ,Pred. TAD

Busulfan

Therapy

Y - Y t(l:3)(q23 25;q29),del(6)(p12), del[11](q12),i(17q],17q +

+ 8 , * 8, + 1 9 , + 22(q-?)/ + 8, + 8. + 19, + 22(q-?),i(17q)

+8,+17,+19,+Ph/ + 8 , + 17,+ 1 9 , + P h , 1 q - / +8,+ 17,+ 19,+Ph,1q-,7q+

+ 8,i(17q)

+ 2 , + 6 , + 9 , + 11, + 1 1 , + 1 2 , + 1 3 , + 13, + 14,+ 1 4 , + 1 8 , + 19, + 1 9 , + 2 0 , * 21, + 21, + P h , t ( 1 ; l l ) ( q 2 1 or 23;p15)

+ 8, - 1 8 / - Y

-Y

Bone marrow

Y / + Ph,t(10:16)(q26;q12),i(17q)

i(17q) dup(17)(q21q25) +19 Y -y

VPh/AI/A1

Ph/A Ph/A A A A A

50/8

42/14 8/8 23 35 46 23

14/1

2(1

A + 18.+ 1 9 , + 2 0 , + P h . + P h A1/A2

2/8/7/4

Ph/A1/A2/A3

+ 3 , + 8, + 8 , + 10, + 1 3 , + 14, + 15,

16

A

1/7/100

N/Ph/A

17 1/62

13/1/16

23/5/9 Ph/A1/A2

Ph/A1/A2

+ 6, + 8 , + 1 3 , + 1 9 , + 1 9 , + Ph, del(20)(qll)

Y

A Ph/A

18/

Distribution of clones/ N u m b e r of metaphases analysed

+8 + 8,i(17q}

Y

+8,

Unstimulated peripheral blood

Karyotype in a d d i t i o n to Ph

Ph chromosome positive patients with additional aberrations found in BC

O

Patient

Sch.R.

D.L.

W.L

L.H-H.

K.H.

B.U.

H.We.

M.H.

Case no.

21

22

23

24

25

26

27

28

Table 2B

12/13/83 02/10/84 02/11/87 04/24/87 09/30/87 01/22/88 05/03/86 07/26/88 11/03/88 04/28/87 05/19/87 08/25/87 10/21/87 03/16/84 09/12/86

M M M M M M M M M M M M M M M

M M

F

04/08/88

12/06/83 05/30/84

F F M M M F F F

Sex

01/12/87 10/12/87 07/08/87 11/04/87 03/16/88 07/15/87 10/15/87 01/12/88

Culture date

57 57 47 47 47 48 48 48 48 45 45 45 45 47 49

57 58

58

51 51 37 37 38 57 57 58

Age

CP BC CP CP CHR CHR AP AP AP CP CP AP BC CP AP

CP AP

AP

CP BC CP AP AP CP CHR CHR

Status of disease

IFNa2b IFNa2b IFNa2b IFNa2b IFNa2b Busulfan IFNo~2b IFNa2b IFNa2b Busulfan Busulfan

Busulfan

Busulfan Hydroxycarbamid

IFN,~2b

IFNa2b IFNa2b

Busulfan Busulfan |FNa2b IFNa2b IFNa2b

Therapy

-7 -7 det[20l(qll)

-7 -7 del(20)(ql 1) del(20)(ql 1) del{20)(ql 1) deI(z0)(q11) del{20)/q11) del(20){q11] del(20}(q11} Y Y Y Y

- Y/inv[l 7)[p13q11) inv(17)(p13q11)

dei120)lq111 del(2O](q11) del(2O)(q11)

17,t[7;17)(p11:qll)/ + 8, + 19,i(17q) - 17,t(7:17)(p11;q111/ + 8, + 19, + Ph,i/17q} +21 i(17q)

del(7)(p13)/+ 8

+ ( 4 o. 5 ) , + 8 , + 1 7 , + 1 9

17,t[7;17){p11;q11)/ + 8 , + 19,i(17q) - 17,t(7;17)(pl 1 ;q] 1)/ + 8, + 19, + Ph,i(17q} +21

+8 +8

+ ( 4 o. 5 ) , + 8 , + 1 7 , + 19

Bone m a r r o w

Unstimulated peripheral blood

Karyotype in a d d i t i o n to Ph

10 4/20 6/51 3O 10/41/4 4 15 20/22/24 11/9/56 31/72 3/1 7/59 10/9 88 19 32 14 1/24 2O 27 100/19 86/25 124/3 146/1 20/1 20

Ph/A1/A2 Ph/A Ph/A N/A N/A A A A A N/A A A Ph/A Ph/A Ph/A Ph/A A1/A2 A

D i s t r i b u t i o n of c l o n e s / N u m b e r of m e t a p h a s e s analysed Ph Ph/A N/Ph A Ph/A1/A2 Ph Ph Ph/A1/A2

Ph chromosome positive patients with additional aberrations found in CP with progression to AP or BC

{.O

F.Ch.

Sch.K T.F.

S.R.

Sch.M.

B.G.

B.V.

L.M.

R.T.

M.B.

CR. W.R H.Wa.

S.A Z.L. D.K. K.M.

H.B.

29

30 31

32

33

34

35 36 37

38

39

40 41 42

43 44 45 46

47

L.J.

Patient

Case UO,

Table 2C

M F M M M M M M M

F

10/02/87

F

06/22/88

01/30/85 04/19/85 12/03/87 02/16/88 05/27188 02/06/87 05/08/84 01/27/88 02/21/89

M M M M F F

03/25/88 04/23/86 11/25/87 02/24/88 08/26/87 03/15/88

M M M F F M M M M M F F F

F F F M

04/23/86 10/01/86 06/16/88 03/23/88

05126187 04/28188 11/09/88 04/23/85 06/12/85 04/19/88 05/25/88 08/24/88 10/23/86 11/27/86 06/16/87 01/27/88 04/29/88

Sex

Culture date

31

17 38 76 77 77 31 14 52 36

51 52 52 47 43 40 40 40 52 52 45 46 46

32

63 34 35 36 31 32

19 19 56 63

Age

CP CP CP CP CP CP CP CP CP CP CP CP Status after B M T CP CP CP CP CP CP CP CP relapse after BMT CP

CP

CP CP CP CP CP CP

CP CP CP CP

Status of disease

Busulfan

Busulfan -IFNa2b

Busulfan IFNc~2b

-Busulfan

1FNa2b

BusuKan IFNct2b IFNc~2b Busulfan Busulfan -Busulfan IFNa2b

IFNa2b

IFNa2b Busulfan IFNa2b IFNa2b IFNa2 b IFNa2b

IFNa2b IFNa2b Busulfan IFNa2b

Therapy

Ph Ph/del(7)lpt 5) Ph/del(7)[pt 5) 7 8 8 8 8

+X

-Y -Y

±8,+19 + Ph + Ph * Ph

* 8 ~8

+ + + + + +

t[2;15)(qll;q15) t{2;15)(q11;q15) Iq + dup(1)(q23~q32)/ t(1;5)(q2l or 22:q31] dup(1)(q23~q32) t(1;5]{q21 or 22:q31]

t(1;17)(q31 or 32:p11) t(2;15)(p23 or 25;q15), t(3;17)(p21:p11)

Bone marrow

+X

Y del(13)(q12.1q22)

-y

+8

+8

+8

+ Ph/del(7)(pl 5) +Ph/del(7)[p15)

t(2:15}(q11;q15) t(2:151(q11;q15)

t(2;15}(p23/25;pl 5),t(3;17)(p21;p11)

t(1 ;17)(q31/32:p11)

- 8,t(8:17)(q11;q25)

Unstimulated peripheral blood

Karyolype in a d d i t i o n to Ph

Ph chromosome positive patients with additional aberrations found in CP without progression

A*

N/Ph/A Ph/A Ph/A P"~IA Ph Ph/A Ph/A Pb/A A

PI~/A PhiAliA2 Ph/A1/A2 Ph/A Ph/A Ph/A A N/Pb/A Ph Ph/A Ph/A Ph/A

N/Ph/A1/A2

Ph/A Ph N/Ph/A N/Ph/A N/Ph/A N/Ph/A1/A2

Pfl Ph/A A Ph/A

17

4/19/5 32/6 37/2 6714 14 262 59/2 1/z3 30

5121 26/7/6 1312/8 19/2 15/5 14/10 24 2/12/11 23 14/1 23/2 35/13

6/10/11/3

24/1 44 6/15/11 2/12/34 3/45/21 5/12/5/3

6 19/3 34 24/1

Distribution o[ clones/ N u m b e r of m e t a p h a s e s analysed

t',,a t'~

203

Cytogenetic Peculiarities in CML

1

2

ii tj 6

7

4

3

• iI'

11t

9A

8

10

++ 13

14

15

Vt 19

20

21

5

22

11

12

!i

..'

++

16

17

18

-¢ XX

Figure 1 Karyotype of a bone marrow cell from patient W. I. (case 23). Arrowhead, t(9;22) leading to 9q+ and 22q- (Ph); open arrow, t(7;17) leading to a monosomy 7p and 17p.

c h r o m o s o m e s 7 and 17 resulting from a translocation (7;17)(p11;q11) (Fig. 1) besides other clones. Patient M. H. (case 28) had two clones, one with loss of the Y chromosome and the other with a pericentric inversion of chromosome 17 in CP (Fig. 2A).

Additional aberrations found in CP without progression to BC (Table 2C): Eighteen patients, whose data are s u m m a r i z e d in Table 2C, belong to this group. Until now only one of them (F. Ch., case 29) progressed to BC, but was not studied at the time. Figure 2B shows a translocation (8;17)(q11;q25) leading to a partial m o n o s o m y 8 that was found in patient F. Ch. (case 29) in CP 13 months after the initial diagnosis of CML. Seven months later the disease had progressed and F. Ch. died in BC 17 months after the last cytogenetic analysis. Two further aberrations involving chromosome 17 were found in patients Sch. K. and T. F. (cases 30 and 31) (Figs. 2C and D). A translocation (1;17) was found in patient Sch. K. (case 30) in June of 1988, at the time of the initial diagnosis of CML in CP. The patient is still in CP. A translocation (3;17) was detected in the bone marrow of patient T. F. (case 31) in CP. Moreover, this patient showed a translocation (2;15)(p23 or 25;q15) within the same clone (not demonstrated in Figure 2).

204

F. Hild and C. Fonatsch

a

inv(17)(p13q11)

t(1;17)(q31;p11)

C

180 °

17

1 t (8;17)(q11;q 25)

1 8

Q

17 t(3;17)(p21;p11)

r

i

rj 1 t

3

d

]| ,t m 17

Figure 2 Partial G-banded karyotype of marrow and unstimulated peripheral blood cells and schematic presentation of four aberrations involving chromosome 17: (a) patient M. H. (case 28), inv(17/; (b) patient F. Ch. (Case 29), t(8;17); (c) patient Sch. K. (case 30), t(1;17); (d) patient T. F. (case 31), t(3;17).

Another abnormality involving chromosomal region 1 5 q 1 5 - - n a m e l y an insertion of 15q15-qter in the long arm of chromosome 2 - - w a s seen in patient S. R. (case 32) during CP (Fig. 3). Figure 4 demonstrates a duplication with inversion of the long arm of chromosome 1 and a translocation (1;5)(q21 or 22;q31) that were found as a mosaicism in addition to normal cells and cells carrying only the Ph translocation in two different clones during the CP of patient Sch. M. (case 33). Patient B. G. (case 34) with a constitutive pericentric inversion of chrcJmosome 9 showed a second Ph chromosome u n d e r therapy with busulfan and interferon (IFN) at first diagnosis of CML, and 11 months later, still in the CP, developed a second clone with a partial deletion of the short arm of chromosome 7, del(7)(p15) (Fig. 5). Patient K. M. (case 46) has been analyzed cytogenetically for the first time in our laboratory, w h e n he was in relapse, 7 years after bone marrow transplantation (BMT).

205

Cytogenetic Peculiarities in CML

(! 1

2

3

4

5

A 6

7

8

9 e

61

10

11

12

aa

:A

64

16

17

18

A 13 oe

19

14 t!

20

15 eo

21

" " A 22

Ir

XY

Figure 3 Karyotype of a Ph positive bone marrow cell from patient S. R. (case 32) with an insertion of chromosome 15 in the long arm of chromosome 2, t(2;15)(q11;q15). Arrowheads indicate the marker chromosomes.

He presented an interstitial deletion of chromosome 13, del(13)(q12.1q22), in a d d i t i o n to the Ph translocation (Fig. 6).

Ph Negative Patients (Table 3) All 17 Ph-negative CML patients studied had normal constitutional karyotypes except for patient G. H. (case 55) who had been examined during blast crisis of the disease, and showed the constitution XYY. Seven patients, all males, had a normal karyotype (cases 48-54), although two of them (B. J. and M. J., cases 52 and 54, respectively) were in blastic phase. Out of the remaining 9 patients who showed karyotypic abnormalities, 8 were in the stable phase of CML. The results of patients J. O. and M. E. (cases 57 and 62) have been published previously [29]. M o n o s o m y 7 was found in two cases, in patients S. S. and S. K. (cases 58 and 59) being in the CP and BC, respectively. Trisomy 8 was found in four patients (cases 60-63). A translocation (4;16)(qll or 12;p13) was detected in the bone marrow and u n s t i m u l a t e d blood cells of patient N. R. (case 56).

Patient

O.S. Z.W. G.E-F. G.F.

B.J.

S.H. M.I.

G.H.

N.R.

J.O. S.S. S.K. G.Ch, T.E. M.E. B.R.

K.H.

48 49 50 51

52 53 54

55

56

57 58 59 60 61 62 83

64

01/22/86 11/17187 01/12/87 06/01/88 04/12/88 05122186 04/17/86 07/11/86 10122/87

01!06/89

10/03/85 11/26/84 02/19/88 07/15/86 05/15/87 02/02/88 11/27/84 04128/88 04/29/88 12/01/87 02/22/88 11/09/88

Cullure date

M

F M M M F F M

M

M

M M

M

M M M M

Sex

54 53 79 34 73 24 40 49 60

34

47 84 69 64 65 42 31 7{5 76 66 67 34

Age

CP CP BC CP CP CP CP CP CP

CP (E{}}

CP CP CP CP CP BC CP (Eo} BC BC BC BC CP (Eo)

Status {}f disease

Hvdroxycarbamid

----

Prednison

IFN

Busulfan Busulfan I lydroxy{;arbamid

Busulfan

Busulfan

Busulfan Busulfan

Therapy

List of Ph c h r o m o s o m e negative patients

Case no.

Table 3

46,XY/49,XY, + 8, + 10, + 15,18q + 46,XY, - 8, - 19, + mar. + mar

46,XY/47,XY, + 8 47,XX, + 8 47,XX, + 8,5q

46,XY 46,XY 46.XY 47,XYY 47,XYY 40,XY/46.XY.I[4:16}{q I 1 or 12;p13) 46,XY/46,XY,t[4;16)(tll I or 12;p13) 46,XX,t{6;9}{p23;q84}

46.XY 46,XY 46,XY

~Olle lllilrrogv

46,XY 49,XY, + 8, + 10, + 15,18q +

47,XX, + 8 47,XX, + 8,5q 46,XY

46,XX,t(6;9)(p23;q34) 45,XY, - 7 46,XY/46,XXY, 7

4~i,XY 4~LXY 47,XYY 47,XYY 46,XY.l(4;16){ql 1 or 12;p13)

46,XY 46,XY 46,XY 40,XY 4{LXY 4{i,XY

Unstimuhlted peripheral blood

Karyolvpe in addition to Ph

A A N/A N/A A A N N/A N/A

N'A

N N N N N N N N N A A NiA

65 31 26/6 24/1 48 79 27 21/7 53/3

4128

29 30 28 5 15 24 40 20 24 25 43 1716

Distribution {1[ c l o n e s / N u m b e r of metap]laS{!S analysed

t',o c3~

207

Cytogenetic Peculiarities in CML

dup inv(1 )(q22q32)

DIS'IIUIIIt2011 el" ~

~,XX(4~) ~,XX (~04)

I

l

xif

~,XX (~04)

t (9;22.)

m

~)

t(9;~

(9;22) (33~)

(49~)

t(1,5)(q21/22;q31) up l.n~ (q22q~2) ~qp ~ly (i) (q2~l: 2) (2O4)

i

, t

(37%}

Ill (q22q32)

)¢* At

~)

l ~ l ~ t '87

t(l;5: (q2.1/~ ;c 31) (1R) March '88

t{l;5]

1/22;¢ 31) (104) ,June ' ~

Figure 4

Chromosome I aberrations, dup inv(1)(q22-~q32) and t(1;5)(q21 or 22;q31), and their schematic presentation found in patient Sch. M. (case 33) in two different clones as a mosaicism with normal cells and simple Ph positive cells. At the right the distribution of these clones at different time of investigation is indicated.

DISCUSSION

Variant Ph Translocations The first variant Ph translocation was described by Hayata et al. in 1973 [9]. Since then more than 308 variants have been d o c u m e n t e d [31-33]. The frequency of 4.2% of Ph variants in our investigation is in good agreement with the results of previous studies showing frequencies between 2.3% and 9.6% with a mean range of 4.3% (31, 34I. Except for the Y chromosome, all chromosomes have been found to be involved in variant translocations. The breakpoints found in our patients with variant translocations c o r r e s p o n d well with those cited in previous studies [10, 12, 15]. Regions 2q37, 7q22, and 11q13, w h i c h were involved in translocations in patients B. M., R. H-P., G. K. and H. P. (cases I and 3-5), belong to a group of n o n r a n d o m l y clustered breakpoints. In patient H. L. (case 2), however, a breakpoint 2p15 was found that has not yet been m e n t i o n e d in the catalog of chromosome aberrations in cancer [33]. Furthermore, the structural aberration t(lO;16)(q26;q12) found together with a second Ph and an i(17q) in a d d i t i o n to the variant translocation during BC of patient R. H-P. (case 3) has been described only once as a secondary chromosomal abnormality in CML [35]. It is generally accepted that Ph positive CML patients showing unusual or c o m p l e x translocations do not differ clinically, hematologically, or prognostically from CML

208

F. Hild and C. Fonatsch

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patients with a standard Ph translocation [36]. However, in a recent review of Phpositive CML patients who had been treated with bone marrow transplantation (BMT) in the accelerated phase, Przepiorka and Thomas found an increased risk of relapse among all those patients who had a variant Ph translocation as compared to patients showing the standard translocation without additional aberrations [37]. For final prognostic conclusions, however, a greater n u m b e r of consecutive cytogenetic studies of patients with identical variant translocations is needed.

Additional Chromosomal Abnormalities (ACA) in CML The occurrence of secondary abnormalities in the acute phase of CML is well established. In our study, 73.3% of the Ph-positive patients in the AP or BC presented additional aberrations, which is in agreement with previously published data [17-19, 22, 23, 29, 32, 34-36, 38-47). Persistent secondary changes in the chronic phase, however, are rarer and less well understood. In contrast to the results of other authors, as for instance of Bernstein et al. [18], G6dde-Salz et al. [19], Sokal et al. [24], who reported frequencies of 9% and 8.8%, respectively, a significantly higher rate, namely 19% (23/120), of additional aberrations in the CP was found in our investigation. Krulik et al. [22] observed ACA during the CP in 5 out of 28 Ph positive patients (17.7%) but they did not m e n t i o n a single case of ACA at the time of the initial

209

Cytogenetic Peculiarities in CML

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Figure 6 Karyotype of an unstimulated peripheral blood cell from patient K. M. (case 46). Arrowheads, t(9;22); open arrow, interstitial deletion of chromosome 13 del(13)(q12.1q22).

diagnosis of CML. In our investigation, 8 of the 23 patients (34.8%) with ACA in the CP showed ACA already at the time of the initial diagnosis. Certain n o n r a n d o m karyotypic changes represent frequent additional secondary anomalies in the blastic phase of CML but also in some cases in the chronic phase of CML, as for instance trisomy 8, isochromosome 17q, a second Ph chromosome, trisomy 19, and loss of the Y chromosome. All these well-established anomalies were present in 30 of our patients (16 in BC, 14 in CP), both as single anomalies and in association with additional numerical or structural aberrations. However, besides these w e l l - k n o w n chromosome anomalies, a n u m b e r of new and interesting structural aberrations were found in our CML patients. Whereas Sessarego et al. [23] described a high rate of secondary structural chromosome aberrations in the BC, we detected additional structural abnormalities, especially in the CP, which has been considered a rather rare event [21, 44]. Additional aberrations involving chromosome #17: Particularly remarkable is the frequent participation of chromosome 17 in structural aberrations. Six out of 40 patients (15%) with additional anomalies showed structural changes of chromosome 17 other than i(17q). Borgstr~Sm et al. have u n d e r l i n e d the n o n r a n d o m occurrence of aberrations for chromosome 17 in myeloproliferative disorders (48). Apart from the loss or gain of a chromosome 17 and the well-known i(17q), they detected structural

210

F. Hild and C. Fonatsch anomalies of c h r o m o s o m e 17 in 5 Ph positive and in 1 Ph negative CML patient as well as in 4 ANLL and preleukemia patients. As other authors, they assumed the structural anomalies of chromosome 17, especially those involving part of or the total short arm, to represent the crucial event in the d e v e l o p m e n t of the terminal phase of myeloproliferative diseases. However, several of our patients d e v e l o p e d chromosome 17 aberrations during a stable and long lasting CP. Most of the structural chromosome 17 anomalies seen in our patients have not yet been described in this particular form: Patient D. R. (case 17), who was only once analysed in the AP, presented a dup(17)(q21-~q25). Patient M. H. (case 28) showed a pericentric inversion, inv(17)(p13q11), that was first detected in the CP in 1984. At that time a second clone existed marked by a missing Y chromosome. The disease was stable during 2 years under therapy with Busulfan. The second analysis in 1986, when the AP began, showed that the clone with inv(17) had s u p p r e s s e d the clone with the missing Y. In patient W. I. (case 23) a translocation (7;17}(p11;q11) was detected as a secondary anomaly occurring in 37% of the metaphases analysed in the CP while two other clones showed only a Ph or a Ph, + 8, ÷ 9 and an i s o c h r o m o s o m e 17q. Five months later in the AP, the t(7;17) clone d i m i n i s h e d to 11% of the metaphases analysed and the i(17q) clone dominated. The patient died in the BC 6 months after the first detection of the clones with secondary anomalies. A similar translocation has recently been described by Tien et al. in a case of Ph positive CML although in the BC [35]. In patient F. Ch. (case 29) a translocation (8;17)(q11;q25) was found 7 months prior to the onset of the BC. Four cases of t(8;17) in CML have been mentioned in the literature. They showed, however, different breakpoints and no partial m o n o s o m y 8 [39, 46, 49]. A translocation (1;17)(q31 or 32;p11) was detected in patient Sch. K. (case 30) at the time of initial diagnosis of CML. This type of translocation has been described in 9 different CML patients who were already in the BC. Seven of these cases showed a partial trisomy 1 due to translocation or nondisjunction [35, 39, 48, 50-52]. In the remaining 2 cases other breakpoints were involved in the trans]ocation (1;17) than in our case [39, 47]. Patient T. F. (case 31) showed a t(3;17)(p21;p11). An identical translocation has been described by Gyger et al. [53] in a case of MDS and by Benitez et al. [54] in a case of ANLL (FAB M5) but has not yet been observed in CML. In another 7 cases the t(3;17) although with different breakpoints, has been interpreted as an aggravating side effect of myeloproliferative disease [23, 55-58]. Our patient T. F. has r e m a i n e d in CP since the diagnosis was established, although he showed an additional t(2;15)(p23 or 25;q15) in the same clone. A t(2;15) was found in another of our patients (S. R., case 32] who did not show s y m p t o m s of acceleration in his CML either. Translocations involving chromosomes 2 and 15 have hitherto not been described in CML.

Additional aberrations involving chromosome 1: In contrast to chromosome anomalies that are considered to be of some specificity for certain malignant diseases, structural changes of chromosome 1 occur in almost all malignant tumors. Evidently CML is not different in this context. In addition to the already k n o w n c h r o m o s o m e 1 translocations [33], we can add the following new types that have not yet been described: The t(1;17) was already m e n t i o n e d above (case 30).

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A t(1;11)(q21 or 23;p15) occurred during the e x t r a m e d u l l a r y progress of the BC of patient M. S. (case 9) together with several numerical anomalies [29]. A translocation (1;11) with different breakpoints has been observed by Hagemeijer et al. [59] in a CML patient presenting in the BC. A t(1;3)(q23 or 25;q29) was found in patient P. R. (case 20) in the BC, together with other structural anomalies of chromosomes 6 [del(6)(p12)], and 11 [del(11)(q12)], as well as an i(17q) and a 1 7 q + . Patient T. H. (case 13) showed an l q from a translocation (1;17).

and a 7q+ in the BC that probably resulted

A d u p l i c a t i o n with simultaneous inversion of 1q23q32 was detected in patient Sch. M. (case 33) in mosaicism with a translocation (1;5)(q21 or 22;q31). Duplication of c h r o m o s o m e 1 is often encountered in CML, involving either the long [21, 33, 60] or short arm [45, 61]. A simultaneous inversion of the d u p l i c a t e d l q segment as well as a t(1;5) have, however, not yet been observed. It is remarkable that both anomalies occurred in the CP of patient Sch. M. without any indication of influencing the course of the disease.

Additional aberrations involving chromosome 7: Monosomy 7 as detected in patients B. V. (case 35) and K. H. (case 25) is very often found in Ph positive CML either as single a d d i t i o n a l aberration or a c c o m p a n i e d by other aberrations [33]. Partial m o n o s o m y for the long arm of chromosome 7 is k n o w n as well [10, 62, 63]. A deletion of the short arm of c h r o m o s o m e 7 has as yet only been described twice in Ph positive CML and once in Ph negative CML in the BC [18, 35, 64]. Three of our patients showed such a partial m o n o s o m y 7p. Patient W. I. (case 23) had a loss of 7p11-pter, in connection with the translocation (7;17) described above. Patient B. G. (case 34) d e v e l o p e d a del(7)(p15). Patient D. L. (case 22) showed a del(7)(p13). All patients d e v e l o p e d the clone with 7p aberrations during interferon (IFN) therapy, besides other aberrations. The possibility of an induction of these secondary anomalies by the IFN therapy has to be taken into consideration and careful further investigation should be carried out to elucidate this assumption.

Additional aberrations involving chromosome 20: Loss of material of the long arm of c h r o m o s o m e 2 0 - - d e l ( 2 0 ) ( q 1 1 ) - - i s accepted as one of the characteristic anomalies of p o l y c y t h e m i a vera [65]. Nevertheless, this anomaly is also found in other myeloproliferative and m y e l o d y s p l a s t i c syndromes. It has been described in 7 cases of CML [35, 40, 66-70]. A del(20)(q11) occurred in one of our patients (B. U. case 26) as the only additional aberration to the Ph translocation at the time of the initial diagnosis of CML. In the other patient (K. L. case 12), del(20q) was found together with n u m e r i c a l aberrations and an additional Ph in the BC. In the latter case the cytogenetic data prior to the BC were unfortunately not available. Additional aberration in a case of leukemic relapse after BMT: The cytogenetic findings in Patient K. M. (case 46) gave rise to a particular question: he d e v e l o p d a relapse of his disease 7 years after bone marrow transplantation, the donor being his brother. The relapse was cytogenetically manifested by a reappearance of the Ph c h r o m o s o m e and by the presence of a del(13)(q12.1q22) in all metaphases analysed. The interstitial deletion in the long arm of chromosome 13 had not been present prior to BMT, according to information provided by the laboratory that had p r e a n a l y z e d the

212

F. Hild and C. Fonatsch patient (Becher, personal communication). The recurrence of Ph positive leukemia in donor cells after BMT has recently been described by Marmont et al. [71]. Since bone marrow samples from K. M.'s brother were not available for comparison of fluorescence p o l y m o r p h i s m s , the question cannot be answered whether the n e w l y appearing c h r o m o s o m e anomaly and thus the relapse of the disease were developing in the donor's cells or resulted from minimal disease of the host. A controversial discussion concerning the significance of additional anomalies at different stages of CML is still going on. For a long time the appearance of additional anomalies was considered as heralding aggravation of the disease. It is now well established that 75% to 90% of the patients in AP or BC of CML show additional anomalies. Whereas some authors believe that additional chromosomal anomalies in the BC do not significantly influence the chance of survival from a clinical point of view [17, 18, 23, 38], other authors indicate that secondary anomalies in CML coincide with a reduction in survival time [43, 45]. But how do things stand with the anomalies that appear early in the chronic phase or exist already at time of the initial diagnosis of CML? Krulik et al. [22] evaluated this question in a consecutive cytogenetic study of 28 patients and c o n c l u d e d that additional anomalies that appear not more than 6 months prior to the onset of BC are of prognostic significance in announcing an aggravation of the disease, but that those anomalies that occur earlier in the disease or are present already at the time of initial diagnosis do not reflect on the prognosis or progress of the disease. On the other hand, Sokal et al. [27] concluded that additional anomalies are an i n d e p e n d e n t factor and are a poor prognostic index with respect to the progress of the disease. They found that patients who showed additional anomalies in the CP at the time of the initial diagnosis did not behave differently during the first 2 years as c o m p a r e d to other CML patients as far as the survival time was considered [24]. However, after 2 years, the mortality rate was 40% higher than that of those patients without additional aberrations at the time of the initial diagnosis. In their study on Ph positive CML patients who received BMT in the AP or BC, Przepiorka et al. [37] showed that specific cytogenetic changes, for instance variant Ph translocations or secondary anomalies like trisomy 8 and duplication of Ph, do represent a bad prognosis. Patients showing these anomalies prior to the BMT suffered from a higher relapse rate than patients without or with other secondary anomalies. Whether these results can be transferred to other therapy forms or to other specific chromosomal aberrations in CP has still to be clarified.

Ph Negative CML Five to ten percent of all CML cases are Ph negative [4, 6, 42]. Lack of a Ph is associated with a significantly lower m e d i a n survival time of about 8 to 20 months as c o m p a r e d to 30 to 55 months in Ph positive CML [5]. However, the so-called Ph negative CML was shown to constitute a heterogeneous group of prognostically distinct disorders w h i c h ultimately will have to be differentiated cytogenetically and otherwise [8, 73]. About 30% of the Ph negative CML patients display chromosome aberrations [4-8, 42]. Nine out of the 17 (52,9%) CML patients found to be Ph negative in our study showed numerical and/or structural chromosome.abnormalities (cases 56-64) not including case 55 (G. H.) with a constitutional supernumerary Y. Some n o n r a n d o m but not disease-specific chromosome anomalies are found in Ph negative CML such as trisomy 8 (as in 4 of our patients, cases 60-64), m o n o s o m y 7 (as in 2 of our patients, cases 58 and 59), aberrations of chromosome 5 (as in the 5 q - found in patients M. E., case 62), trisomy 21, loss of Y and rarely an i(17q) [5, 33]. The three latter anomalies were not present in our patients. Translocations are also not u n c o m m o n in Ph

Cytogenetic Peculiarities in CML

213

negative CML, those involving chromosome 3 being the most common, followed by translocations involving chromosomes 6, 9, and 11 [33]. Among our patients with Ph negative CML, 2 presented with translocations: Patient J. O. (case 57) showed a t(6;9)(p23;q34) that so far has been reported in 42 cases with malignant hematologic diseases, preferentially with acute n o n l y m p h o c y t i c leukemias (ANNL) [29, 33, 74-78]. In Ph negative CML, however, only three cases with t(6;9) have been published, including our own patient [29, 79, 80]. An increased n u m b e r of marrow basophils has been found in some of these cases. A peculiar, never described translocation was found in patient N. R. (case 56), a young man who had been exposed to carcinogenic fumes at work. He d e v e l o p e d a Ph negative CML with extensive hypereosinophilia, showing a t(4;16)(q11 or q12;p13).

CONCLUSION Intensive and careful cytogenetic studies during the course of CML provide some helpful information and conclusions: A high frequency of structural anomalies is found in Ph positive and Ph negative CML besides the w e l b e s t a b l i s h e d secondary abnormalities. Most of the structural anomalies seen in our patients had not been described in CML before. Quite a n u m b e r of these anomalies are already present at the time of the initial diagnosis or early in the chronic phase of the disease. This may be of limited prognostic value. Consecutive cytogenetic studies provide knowledge about the clonal evolution in CML and about the behavior and role of different types of aberrations during progression of the disease. The recognition and identification of the breakpoints found in secondary chromosomal abnormalities allows investigations concerning the role of various protooncogenes and other genes that may be involved in chromosomal rearrangements, for example, the proto-oncogenes SKI, located in 1q21.1-q24, MYCN located in 2p24, KIT located in 4p11-q22, RAL located in 7p22-p15, EGFR located in 7p13p12, and HRAS1 located in 11p15.5; furthermore, the genes for the tumor necrosis factors alpha and beta (TNFA, TNFB respectively) are located in 6p21.3 and the gene for the tumor related protein p53 (TP53) m a p p e d to 17p13.1 [811. All these genes have been m a p p e d to chromosomal regions that are n o n r a n d o m l y involved in a d d i t i o n a l and/or secondary aberrations found in our patients. Further investigations are n e e d e d to examine more precisely the correlations beween additional c h r o m o s o m e aberrations, their prognostic significance, and their importance for the response to different forms of therapy and to clarify the molecular basis of c h r o m o s o m e aberrations besides the Ph translocation in CMI. We wish to thank the members of the following institutions for providing leukemic cells and for their helpful collaboration: Prof. Dr. H. Poliwoda, Priv.-Doz. Dr. M. Freund, Dr. H. Diedrich (Abteilung Hfimatologie/Onkologie, Medizinische Hochschule Hannover), Prof. Dr. V. Diehl, Prof. Dr. B. M6dder, Priv.-Doz. Dr. R. Zankovich, Dr. H. Tesch (Medizinische Universitfitsklinik I, K61n), Prof. Dr. H. Barrels (Medizinische Klinik Sfid, Lfibeck), Priv.-Doz. Dr. H. Bodenstein (Klinikum Minden), Prof. Dr. T. Wagner, Dr. G. Schwieder (Klinik f~r Innere Medizin, Medizinische Universit~t zu L~beck), Prof. Dr. Th. Bflchner (Medizinische Klinik und Poliklinik, Inhere Medizin A, Westf~lische Wilhelms-Universitfit Mfmster), Prof. Dr. W. M. Gallmeier, Dr. R. Zippel (Klinikurn Stadt N~rnberg), Dr. W.-D. Ludwig (Medizinische Klinik und Poliklinik, Universit~tklinikum Steglitz, Freie Ur~iversitfit Berlin), Dr. B. Stollmann (Kinderklinik, Universit~ts- Klinikum Essen), Prof. Dr. R. Hehlmann (Klinikum der Stadt Mannheim), Prof. Dr. F. W.

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Aly (Stadtkrankenhaus Wolfsburg), Prof. Dr. W. Schneider, Priv.-Doz. Dr. C. Aul (Medizinische Klinik und Poliklinik, Universit~t Dfisseldorf), Prof. Dr. A. D. Ho, Dr. H. Meier-Willersen (Medizinische Klinik und Poliklinik V, Ruprecht-Karls-Universit~t Heidelberg). We are very indebted to Ms. R. Konopka, Ms. N. Erbe and Ms. U. Kolbus for skillful technical assistance and Dr. W. Hild for editorial help. This study was supported in part by the Deutsche Krebshiye--Dr. Mildred Scheel-Stiflung (M18/88/FOL).

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