A Recurring Chromosome Rearrangement, dic(16;22), in Acute Nonlymphocytic Leukemia

You-Sheng Li, John Anastasi, Richard A. Larson, Michelle M. Le Beau, James W. Vardiman, and Janet D. Rowley

ABSTRACT: A dicentric chromosome, dic(16;22), resulting in the loss of 16q and 22p was seen in bone marrow cells from two patients with acute myelomonocytic leukemia and one patient with a therapy-related myelodysplastic syndrome that evolved to leukemia. Review of the clinical findings and of the bone marrow morphology failed to reveal any distinctive features in common among these patients. The dic(16;22) may be a new, rare, recurring abnormality associated with malignant myeloid disorders.

INTRODUCTION

More than 100 consis tent chromosome rearrangements have been identif ied in hu- man neoplasia , and the number is increasing rapid ly [1]. The great diversi ty of chromosome changes may reflect the complexi ty of the process of tumorigenesis and progression. Recent work in molecular biology has indica ted that some chro- mosome rearrangements result in structural changes or abnormal expression of pro- tooncogenes or other genes governing cell proliferation and differentiat ion and, thereby, play a fundamenta l role in mal ignant transformation. Many of these chro- mosome changes are uniquely associated with specific subtypes of neoplasia , indi- cating that the genes located at the breakpoints in these rearrangements have im- portant functions restr icted to the cell type involved in the neoplast ic process [2, 3]. Thus, character izat ion of newly discovered recurring chromosome rearrange- ments in human neoplas ia is important in providing more precise cl inical diag- rloses and in identifying addi t ional breakpoints as a focus for molecular cytogenetic analysis. We report herein two cases of acute myelomonocyt ic leukemia FAB type M4 (AMMoL-M4) and one case of therapy-rela ted myelodysplas ia (t-MDS) that evolved to acute leukemia, with an ident ical dicentr ic chromosome involving breaks in the long arm of chromosome 16 and the short arm of chromosome 22. The resultant chromosome contains the short arm of #16 and the long arm of #22; therefore, these mal ignant cells are deficient for most of 16q and 22p.

From the Departments of Medicine and Pathology, University of Chicago, Chicago, IL.

Address requests for reprints to Dr. ]anet D. Rowley, Joint Section of Hematology/Oncol- ogy, Box 420, 5841 S. Maryland Ave., Chicago, IL 60637.

Received April 13, 1988; accepted June 21, 1988.

143

¢O 1988 Elsevier Science Publishing Co., Inc. CancerGenet Cytogenet 35:143 150 (1988) 655 Avenue of the Americas. New York. NY 10010 0165-4608/88/S03.5e

144 v. Li et al.

MATERIALS AND METHODS

For cytogenetic analysis, cells from bone marrow aspirates were cultured for 24 or 48 hours in RPMI-1640 medium supplemented with fetal bovine serum (10%), 10 mM HEPES, 100 U penicillin, and 100 ~g/ml streptomycin. Metaphase cell prepa- ration and chromosome staining using a trypsin-Giemsa banding technique were performed as described previously [4]. Cells from patients were also stained using a modification of the C-banding technique of Sumner [5]. Chromosomal abnormal- ities were described according to the ISCN [6].

RESULTS

Case Reports

Case 1. This 74-year-old white woman presented in June 1986 with a history of a MDS for 1 year, associated with increasing symptoms of anemia and recurring epi- sodes of infection and fever. There was no tymphadenopathy or splenomegaty. She had owned and operated a dry-cleaning business for many years and described extensive exposure to solvents. A brother had died from leukemia at 54 years of age. Initial blood counts are listed in Table 1. A diagnosis of AMMoL-M4 was made from examination of bone marrow and blood. The patient received a short course of gamma interferon, with no improvement, followed by low-dose cytarabine, again without improvement. Later, she required hydroxyurea to control a leukocytosis, primarily of monocytes, that eventually reached 132,000/pA. She died at home in October 1986.

Case 2. A 62-year-old man presented in January 1987 with a 6-week history of easy fatiguability. His past medical history was unremarkable. He was a retired pipe fitter but denied exposure to chemicals or radiation. His physical examination was remarkable only for pallor. His spleen was not palpable. The initial blood count showed a white blood cell count (WBC) of 10,200/~1 with 11% blasts (Table 1). A bone marrow examination confirmed AMMoL. He was treated on a Southwest On- cology Group protocol using low doses of etoposide, adriamycin, cytarabine, and 6- thioguanine given every 2 weeks until remission. He entered complete remission and received two consolidation courses with the same therapy but relapsed in Au- gust 1987. The patient was then treated with standard doses of daunorubicin and cytarabine and again achieved a complete remission. At this time he has recently completed his first consolidation course using high-dose cytarabine.

Case 3. In 1972, this hospital social worker was treated for non-Hodgkin's lym- phoma (NHL) with cyclophosphamide, vincristine, procarbazine, and prednisone (COPP). In 1976, her lymphoma relapsed and she again received COPP. This was followed by maintenance therapy with chlorambucil and prednisone through March 1984. She was a cigarette smoker and, in June 1980, she underwent a lobec- tomy for the complete resection of a lung carcinoma. In April 1984, she was noted to have a macrocytic anemia, and bone marrow examination confirmed the diag- nosis of t-MDS with refractory anemia with excess blasts. No therapy was given, and the anemia and thrombocytopenia progressed. In October 1984, a computed tomography (CT) scan of the thorax showed multiple pulmonary nodules, and a needle biopsy demonstrated recurrent lung cancer; she received etoposide and cis- platinum. In December 1984, the WBC was 27,600/p~] with 27% blasts and 32% monocytes. She died in January 1985, at age 57, from metastatic lung cancer. No

Ta

ble

1

Cli

nic

al

ch

ara

cte

rist

ics

Per

iph

eral

blo

od

C

ase

Age

/ P

rele

uk

emic

no

. se

x

Ex

po

sure

his

tory

p

has

e (m

o)

Dia

gn

osi

s H

b

MC

V

WB

C

Bla

sts

Mo

no

B

aso

Bo

ne

mar

row

(%

) S

urv

ival

P

lts

Cel

lula

rity

B

last

s (m

o)

1 74

/F

Dry

-cle

anin

g

12.0

A

MM

oL

8.

4 11

1 10

.1

0 74

0

155

solv

ents

2

62/M

P

ipef

itte

r 1.

5 A

MM

oL

6.

7 --

10

.2

11

22

--

264

3 5

7/F

C

ycl

op

ho

sph

amid

e,

9.0

t-M

DS

11

.8

107

4.2

3 4

13

151

chlo

ram

bu

cil,

v

incr

isti

ne,

p

roca

rbaz

ine,

p

red

nis

on

e (f

or

NH

L)

65

33

5

90

62

12 +

40

17

10

Abb

revi

atio

ns:

Hb,

hem

oglo

bin;

MC

V. m

ean

corp

uscu

lar

volu

me;

WB

C, w

hite

blo

od c

ell

coun

t; M

ono,

mon

ocyt

es;

Bas

o, b

asop

hils

; Pi

ts,

plat

elet

s.

{.71

146 Y. Li et al.

chemotherapy was given specifically for her therapy-related leukemia, and this dis- ease as well as the lung cancer progressed despi te e toposide and c isp la t inum treat- ment.

Pathology Results

Bone marrow aspirates and biopsies that were obtained prior to therapy for leuke- mia and s imul taneous ly with specimens for cytogenetic studies were reviewed. All cases exhibi ted dysplast ic features in the granulocytic series and, to a lesser extent, in the erythroid cell lines. The dysplas ia was evident as hypogranular i ty and ab- normal nuclear lobu]ation, inc luding hypolobula t ion in mature granulocytes, and as nuclear budding and megaloblastoid change in erythroid precursors. The dyspla- sia was mild to moderate as judged by the criteria of Juneja et al. [7].

In addi t ion to the dysplasia, each case exhibi ted a monocyt ic component to the leukemic process. Cases 1 and 2 each met the FAB criteria for AMMoL-M4, but were somewhat different in terms of the number and character of the monocyt ic cells. In case 1, monocytes accounted for 51% of the bone marrow elements and were 7,470/~1 in the per iphera l blood. Despite the unequivocal monocyt ic mor- phology of these cells, however, a lpha-naphthyl acetate esterase (ANA) activi ty was surpr is ingly weak or absent. In case 2, monocytes accounted for 10% of the bone marrow elements and were 2,240/~1 in the per ipheral blood. The ANA activi ty was strongly posi t ive in 20% of the nonerythroid bone marrow cells. The leukemic transformation in case 3, which was evidenced by a per ipheral blast count of 27%, was accompanied by a monocytosis Of 8,800/~1. Further s tudy and subclassif icat ion was not possible because a bone marrow biopsy was not obtained and a per ipheral blood smear was not available for our review.

Case 3 was ini t ia l ly diagnosed as refractory anemia with excess blasts and was dist inct from the other two cases in that the dysplas ia was evident not only in the granulocyt ic and erythroid cell lines, but also in megakaryocytes, eosinophi ls , and basophils . Megakaryocytes were hypolobated and atypical appearing, whi le eosin- ophi ls and basophi ls exhibi ted atypical granulation. Case 3 was also dist inct in that there was a per ipheral blood and bone marrow basophi l ia (13% and 4%, respec- tively); this was not present in the other cases.

Cytogenetic Results

Among 645 patients wi th ANLL de novo, 136 patients with therapy-rela ted acute non lymphocyt ic leukemia (ANLL), and 345 patients with an MDS who had ade- quate material for cytogenetic studies in our laboratory, three patients had a dicen- tric chromosome result ing from a break in the long arm of chromosome 16 at band q11, and a break in the short arm of chromosome 22, at band p l l [(dic(16;22)(q11;p11)], (Figs. 1 and 2). The results of cytogenetic analysis of bone marrow from these three patients is l is ted in Table 2. Each of these patients had a mixture of normal and abnormal mitot ic cells; one patient (case 1) had the dic(16;22) as the sole karyotypic abnormali ty. The remaining two patients had ad- di t ional chromosomal abnormali t ies; an extra chromosome 22 was present in one patient (case 2), and abnormali t ies involving both chromosome 12 homologs and a loss of the homologous chromosome 22 were observed in the remaining patient (case 3).

Analysis of C-banded metaphase cells from each case confirmed the presence of two centromeres in the rearranged chromosome (Fig. 1, inset). The dic(16;22) re- suits in the loss of chromosomal material from the long arm of one chromosome 16 homolog as well as loss of the short arm of one chromosome 22 homolog.

dic(16;22) in ANLL 1 4 7

1 2

i! i( l i 3 4 5

6 7 8 9 10 11 12

t l t lit - .: 13 14 15 17 18

IL

, ~ I | l

19 20 22 Y

16 ~,

i

21

Figure 1 Karyotype of a trypsin-Giemsa banded metaphase cell obtained from a bone mar- row aspirate from case 1 illustrating the dic(16;22). The rearranged chromosome is shown at the right of the chromosome 16 homologs (identified with arrow). Inset: Chromosome 16 and 22 homologs that were sequentially stained using a G-banding technique followed by C band- ing. The presence of two constitutive heterochromatin bands (C bands) in the rearranged chro- mosome confirms the presence of two centromeres.

DISCUSSION

In this report, we describe three pat ients who had ANLL associated with an appar- ently ident ical chromosome rearrangement. Al though we were unable to identify any unique morphologic features associated with this abnormali ty, a few points require further comment . All three patients were older adults, and two of them had had exposure to mutagenic agents. Two of them had a myelodysplas t ic phase that progressed to overt leukemia. The leukemic process showed a monocyt ic compo- nent in each case and dysplast ic features in the granulocytic series and, to a lesser extent, in the erythroid cells. This suggests that the dic(16;22) occurred in a pluri- potent cell that had the capaci ty to differentiate to mul t ip le cell lineages. In some patients who have ANLL, the leukemic process apparent ly involves mul t ip le cell lineages, al though one cell l ineage may predominate in the bone marrow [8-10]. Such patients usual ly are elderly and often have a history of exposure to potent ia l ly carcinogenic/mutagenic agents, especial ly chemotherapy and radiotherapy. Typi- cally, they have a myelodysplas t ic phase prior to the deve lopment of acute leuke- mia. Several recurring chromosome rearrangements already have been described among these patients, and the dic(16;22) may be another.

The diss imilar i t ies among our pat ients regarding their pathologic and cl inical features may reflect the difference in their addi t ional chromosomal changes. Case 1 had the dic(16;22) as the sole abnormali ty; case 2 had t r isomy 22 as well; and case 3 had several other chromosomal changes, inc luding del(12p). This latter chromo- somal abnormal i ty has been associated with an increased number of basophils , a feature noted in the bone marrow aspirate of this pat ient [11]. It is probably signif-

148

c

16 die

Y. Li et al.

Figure 2 Partial trypsin-Giemsa-banded karyotypes illus-

trating the normal chromosome 16 and 22 homologs and the

dic(16;22) chromosome from two metaphase cells from

22 bone marrow aspirates from case 1 (A); case 2 (B); and case

3 ((7.

icant that case 3 showed development of an independent clone in the second bone marrow aspirate sent for cytogenetic analysis. This independent clone contained a deletion of 6q [de1(6)(q16q33)], which we and other investigators have identified as one of the most common features of t-MDS and t-ANLL [12, 131. Although we did not receive later aspirates for chromosome analysis, the presence of the del(6q) and increasing peripheral blood blasts supports the diagnosis of t-ANLL.

Although the patients with ANLL and an abnormality of chromosome 16 that we describe here have a monocytic component to their leukemic process, other features show no similarity, either clinically or morphologically, to those who have an inv(16)(p13q22) or related rearrangements [14]. This is not unexpected, because the breakpoint in 16 involves band qll rather than q22.

Although previously considered to be unstable, dicentric chromosomes have been identified in a number of human tumors. It appears that dicentric chromo- somes may remain stable through multiple cell divisions if the two centromeres are close enough to prevent the formation of anaphase bridges. Alternatively, one of the centromeres may become nonfunctional, thus allowing the two chromatids of the dicentric chromosome to separate synchronously with the other chromosomes in late metaphase [IS].

In summary, this report describes a rare but recurring chromosome abnormality in ANLL. No examples of a dic(16;22) were included in Mitelman’s catalog [16]. Molecular analysis of this rearrangment should result in the identification of those DNA sequences involved in this rearrangement and will provide probes that will

Tab

le 2

R

esu

lts

of c

yto

gen

etic

an

aly

sis

Cas

e N

umbe

r of

cel

ls

no.

Dat

e S

tatu

s T

issu

e an

alyz

ed

Kar

yoty

pe

1 6/

11/8

6 D

iagn

osis

B

M;

D,

24,

and

48

30

2 1/

22/8

7 D

iagn

osis

B

M;

24 a

nd 4

8 23

3 4/

20/8

4 D

iagn

osis

B

M;

24

20

(t-M

DS)

7/

31/8

4 tI

AN

LL

B

M;

24 a

nd 4

8 24

46,X

X(1

4%)/

45,X

X,d

ic(1

6;22

)(q1

1;p1

1) (

83%

)/

SC

A:4

6,X

X, +

21,

dic(

16;2

2) (

3%)

46,X

Y (

83%

)/46

,XY

, +

22,d

ic(1

6;22

)(q1

1;p1

1) (

17%

)

46,X

X (

55

%)/

44

,XX

,- 2

2,de

l(12

)(pl

lp13

),t(

12;?

) (q

l 3;?

),di

c(16

;22)

(q11

;p11

) (4

5%)

46,X

X (

9%)/

44,X

X,

22,d

el(1

2)(p

llp1

3),

t(12

;?)(

q13;

?),d

ic(1

6;22

)(q1

1;p1

1) (

54%

)/

46,X

X,-

16,

del(

5)(q

15q3

3),

+ de

r(16

}t(1

;16)

(q

21;q

24)

(33%

)/S

CA

:45,

XX

, 22

,+16

,de1

(12)

, t(

12;?

),di

c(16

;22)

(4%

)

Abb

revi

atio

ns:

D, d

irec

t, 24

and

48

desi

gnat

e ho

urs

in c

ultu

re;

SCA

, sin

gle

cell

abno

rmal

ity.

ga

150 Y. Li et al.

be use fu l for m o r e p rec i se c l i n i ca l d i agnos i s , as we l l as da ta t ha t wi l l be i m p o r t a n t in the d e v e l o p m e n t of m o r e speci f ic t he rapy .

Supported in part by the U.S. Department of Energy Contract No. DE-FG02-86ER60408; USPHS Grants CA-16910 and CA-42557 (J. D. R.); and University of Chicago Cancer Research Foundation (J. D. R., M. M. L.).

Dr. Larson is the recipient of a Clinical Oncology Career Development Award from the American Cancer Society. Dr. Le Beau is a Scholar of the Leukemia Society of America.

The authors acknowledge the expert technical assistance of Elizabeth M. Davis, Mary Eliz- abeth Neilly, and Britte N. Harris; Dr. Sucha Nand for referring case 2 and Dr. Richard Desser for referring Case 3. Sheila Cotton provided expert secretarial assistance.

REFERENCES

1. Bloomfield CD, Trent IM, van den Berghe H (1988): Report of the committee on structural chromosome changes in neoplasia. Human Gene Mapping 9 (Paris 1987). Cytogenet Cell Genet 46:344-366.

2. Rowley JD (1984): Biological implications of consistent chromosome rearrangements in leukemia and lymphoma. Cancer Res 44:3159-3168.

3. Le Beau MM, Rowley JD (1986): Chromosomal abnormalities in leukemia and lymphoma: Clinical and biological significance. In: Advances in Human Genetics, H Harris, K Hir- schhorn, eds. Plenum Press, NY, pp. 1-54.

4. Le Beau MM (1984): Cytogenetic analysis of hematologic malignancies. In: Association of Cytogenetic Technologists, Cytogenetics Laboratory Manual, M Wahrenburg, ed. pp. 1-93.

5. Sumner AT (1972): A simple technique for demonstrating centric heterochromatin. Exper Cell Res 75:304-306.

6. ISCN (1985): An International System for Human Cytogenetic Nomenclature, DG Harnden, HP Klinger, eds.; published in collaboration with Cytogenet Cell Genet (Karger, Basel; 1985); also in Birth Defects: Original Article Series, vol. 21, No. 1 (March of Dimes Birth Defects Foundation, NY, 1985).

7. Juneja SK, lmbert M, Jouault H, Scoazec J-Y, Sigaux F, Sultan C (1983): Hematological features of primary myelodysplastic syndrome (PMDS) at initial presentation: A study of 118 cases. J Clin Pathol 36:1129-1135.

8. Fialkow PJ, Singer JW, Adamson JW, Vaidya K, Dow LW, Ochs J, Moohr JW (1981): Acute nonlymphocytic leukemia: Heterogeneity of stem cell origin. Blood 57:1068-1073.

9. Raskind WH, Tirumali N, Jacobson R, Singer J, Fialkow P (1984): Evidence for a multistep pathogenesis of a myelodysplastic syndrome. Blood 63:1318-1323.

10. Ferraris AM, Raskind WH, Bjornson BH, Jacobson RJ, Singer IW, Fialkow PJ (1985): Het- erogeneity of B-cell involvement in acute nonlymphocytic leukemia. Blood 66:342-344.

11. Berger R, Bernheim A, Le Coniat M, Vecchione D, Pacot A, Daniel MT, Flandrin G (1986): Abnormalit ies of the short arm of chromosome 12 in acute nonlymphocytic leukemia and dysmyelopoietic syndrome. Cancer Genet Cytogenet 19:281-289.

12. Le Beau MM, Albain KA, Vardiman JW, Larson RA, Blough R, Davis EM, Golomb HM, and Rowley, JD (1986): Clinical and cytogenetic correlations in 63 patients with therapy- related myelodysplastic syndromes and acute nonlymphocytic leukemia: Further evi- dence for characteristic abnormalities of chromosomes 5 and 7. J Clin Oncol 4:325-345.

13. Pedersen-Bjergaard J, Philip P, Pedersen NT, Hou-Jensen K, Svejgaard A, Jensen G, Nissen NI (1984): Acute nonlymphocytic leukemia, preleukemia, and acute myeloproliferative syndrome secondary to treatment of other malignant diseases. Cancer 54:452-463.

14. Larson RA, Williams SF, Le Beau MM, Bitter MA, Vardiman JW, and Rowley JD (1986): Acute myelomonocytic leukemia with abnormal eosinophils and inv(16) or t(16:16) has a favorable prognosis. Blood 68:1242-1249.

15. Vig BK, Zinkowski RP (1986): Sequence of centromere separation: A mechanism for or- derly separation of dicentrics. Cancer Genet Cytogenet 22:347-359.

16. Mitelman F (1988): Catalog of Chromosome Aberrations in Cancer, 3rd Ed. Alan R. Liss, NY.