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Two Distinct Loci on the Short Arm of Chromosome 16 Are Involved
in Myeloid Leukemia
By J.W. Wessels, P. Mollevanger, J.G. Dauwerse, F.H.M. Cluitmans, M.H. Breuning, and G.C. Beverstock
We report a case of acute nonlymphocytic leukemia (ANLL)
M 5 with the characteristic t(8;16)(pll;p13). The breakpoint
in the short arm was regionally localized using nonradioactive in situ hybridization with a series of cosmids of chromosome 16. The results show that a difference exists between
the breakpoint in chromosome 16(p13) in this t(8;16) and the
breakpoint involved in the short arm in the characteristic
inversion 16 (p13;q22)) that occurs in ANLL M4eo. Two
different loci appear to be involved in these chromosomal
rearrangements.
o 1991 by The American Society of Hematology.
T
BM showed predominance of blast cells with the same morphology
WO TYPES of acute nonlymphocyticleukemia (ANLL)
as the chloroma. Blast cells were strongly positive for peroxidase
are known to be associated with rearrangements
and weakly positive for a-naphtylacetate-esterase (ANAE). Signs
involving chromosome 16. Four recurrent abnormalities
of disseminated intravascular coagulation (DIC) were found:
have been reported in ANLL M4e0, as classified by the
Fibrinogen was 1.8 g/L, prothrombin time was 15 seconds (norFrench-American-British (FAB) study group.’ A deletion
mal < 14.5 seconds), and fibrinogen degeneration products were
in the long arm, [de1(16)(q22]),’ a translocation between
increased.
the two chromosome 16 homologues, [t(16;16)(~13;q22)],~,~ Analysis of surface antigens of BM cells after Ficoll gradient
a translocation between chromosomes 8 and 16 [t(8;16)(pll;
separation showed positivity for stem cell markers CD34, HLAp13)],5-’ and a pericentric inversion [inv(l6)(pl3;q22)].’.”.’
DR, myeloid markers CD33 and VIM-2, and myeloid-monocytic
In addition, two other translocations involving breakpoint
marker CD14. The diagnosis of ANLL M5 was made according to
the FAB classification. Complete remission was achieved after a
16(q22) in ANLL M4 have been reported, ie, a t(3;16)(q24;
first treatment with daunorubicin and cytosine arabinoside
q22),I0 and a t(5;l6)(q33;q22).l1 In all these rearrange(ARA-C) according to the EORTC LAM-8 protocol. After a
ments, the breakpoints 16(p13), or 16(q22), or both are
consolidation
course with high-dose ARA-C and amsasacrine, an
involved. In ANLL M5, the reciprocal translocation beallogenic BM transplantation (ABMT) was performed.
tween chromosomes 8 and 16 (t(8;16)(pll;p13)) also occ u r ~ . ‘ ~This
- ’ ~translocation has also been reported in a case
MATERIALS AND METHODS
of ANLL M2I5 and malignant histiocytosis.16We recently
Cytogenetics. A 24-hour unstimulated BM culture was synchrostudied a patient with ANLL M5 whose karyotype was
nized by the method of Webber and Garson.’“ BM was harvested
46,XX,- 16,t(8;16)(pll;p13),+der(l6)[t(11;16)(q13;pl3)],
according to standard techniques. Routine GTG-banding was
ie, a reciprocal translocation between chromosomes 8 and
used.2’ For the combined bandindpainting technique, we per16 and a nonreciprocal translocation between chromoformed the GTG-banding technique as described by Smit et aL2*
somes 11 and 16 (Fig 1). The localization of breakpoint
After the metaphases were karyotyped and photographed, slides
16(p13) in the 8;16 translocation is difficult to determine
were destained with 70% ethanol (twice) for 10 minutes. The same
air-dried slides were then reused for the chromosome painting.
cytogenetically with routine GTG-banding owing to its
Probes. The alphoid repeat probes pHUR195 and pSE16,
faintly staining band. We chose a combination of GTGwhich are specific for the centromeric region of chromosome 16,23,24
banding and an fluorescent in situ hybridization technique
were used. To paint chromosome 16, we used a pool of 166 cosmids
with a pool of chromosome 16 specific cosmids to “paint”
isolated from the DNA of a somatic cell hybrid containing a single
chromosome 16. Subsequently, we used single cosmids from
human chromosome 16 in a mouse background.= The order on the
this pool, ranked along 16p, to map the breakpoints to
short arm of chromosome 16 of the cosmids 36 (D16S79), 41
distinct regions of the short arm of chromosome 16. A
(D16S82), 24 (D16S80), 26 (D16S125), and 218 (D16S246) were
detailed map for the 16(p13) region is currently availobtained from family studies’” and mapping relative to known
as a result of intensive mapping efforts to locate the
breakpoints in 1 6 ~ .Cosmids
*~
40 (D16S257) and H17 (D16S258)
gene for the adult form of polycystic kidney disease (PKD1).
map distal to breakpoint PK 32.1 (Fig 2) but are not precisely
localized. Cosmids cos-a111and cos-5’HVR I (M. Kielman, unpubWe used a number of cosmids from this region to determine
lished observations) are cosmids from the a-globin gene cluster. All
whether the two chromosomal rearrangements, the t(8;16)
and the t(11;16), of our patient have the same breakpoints
or breakpoints very similar to the breakpoint in the inv (16)
(p13;q22) reported by Dauwerse et al.I9
From the Department of Human Genetics, State University HospiCASEREPORT
A 29-year-old woman was admitted to our hospital in May 1989
because of chloroma in a cervical lymph node, which was removed.
She had no complaints, and physical examination showed no
abnormalities. No abnormal cells were noted in either bone
marrow (BM) or peripheral blood (PB). No further treatment was
instituted.
She was readmitted to our hospital in December 1989 with
bruising and pain in the left leg and shoulder. The WBC count was
6.2 x 109/L, the differential count showed 15% blasts, and the
platelet count was 30 x 10y/L.Examination of a biopsy specimen of
Blood, Vol77, No 7 (April I),1991: pp 1555-1559
tal; and the Department of Haematology, Academic Hospital Leiden,
The Netherlands.
Submitted July 16,1990; accepted November 30, 1990.
Supported by the Dutch Kidney Foundation.
Address reprint requests to J.W. Wessels, MD, Department of
Human Genetics, State University of Leiden, Sylvius Laboratory,
Wassenaarseweg 72, 2333 A L Leiden, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I991 by The American Sociew of Hematology.
0006-4971I9117707-0024$3.00/0
1555
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WESSELS ET AL
1556
8
tC8;16)
tCll;16)
11
Fig 1. Partial karyotype of a GTG-banded BM
metaphase.
telomere of the short arm of chromosome 16. To characterize the t(8;16) breakpoint further, we hybridized cosmids 24
and 26, which mapped the breakpoint distal to cosmid 24
and proximal to cosmid 26. This interval could be reduced
by using cosmid 218, which mapped the breakpoint between
cosmid 24 and 218.
these probes were biotinylated by standard nick-translation using
biotin-1 1-dUTP or biotin-16-dUTP (Boehringer Mannheim, German~)?’.~
In situ hybridizntion. pSE16, pHUR195, and the chromosome
16 cosmid library were hybridized according to standard condit i o n ~ . *Single
~
cosmids were simultaneously hybridized with
pHUR195 to facilitate recognition of chromosome 16.’9.M.3’
After
hybridization, all probes were detected using FITC-conjugated
avidin DCS (Vector Laboratories, Burlingame, CA). When necessary, the signals were enhanced with biotinylated goat-anti-avidin
(Vector) followed by another layer of FITC-a~idin.~~
The slides
were mounted with antifade medium containing propidium iodide
( ~ ~ 3 3
Microscop. Hybridized metaphases were examined with either
a Zeiss Axioscop (Zeiss, Oberkochen, Germany) or a Leitz
Aristoplan fluorescence microscope (Leitz, Wetzlar, Germany)
with appropriate filter combinations and photographedwith Scotch
640 ASA film.
DISCUSSION
Chromosome 16 rearrangements in ANLL M4 and M5
appear to be related to two major breakpoints, ie, 16(p13)
and 16(q22). It is of clinical interest that involvement of
both breakpoints appears to influence the clinical course of
these patients. In the inversion 16(p13;q22) and t(16;
16)(p13;q22), both breakpoints are involved. These rearrangements are associated with ANLL M4e0, although
several reports have described other ANLL subtypes with
inversion 16(~13;q22).~~.’~
All these cases showed abnormal
eosinophils. A literature re vie^^.^.'^ suggested that this is a
recurring cytogenetic-clinicopathologic association, which
has a favorable prognosis, especially if treated aggressively
with chemotherapy at the time of diagnosis; however,
Bernard et al.” noted no such correlation.
In both ANLL M4 and M5, the translocation between
chromosomes 8 and 16 exists. The prognosis of the t(8;
16)(pll;p13) associated with ANLL is uncertain because
no multiple series of patients with this abnormality has
been reported. To our knowledge, the additional translocation in our patient has never been reported before and at
present we cannot predict whether it has any prognostic
value.
Localizing the breakpoints in chromosome 16 precisely
by standard cytogenetic analysis is often very difficult owing
to contracted chromosome quality or faint banding. The
breakpoint in the short arm of chromosome 16 in ANLL
M4eo and inv 16(p13;q22) was recently localized regionally
by Dauwerse et al.I9 This was achieved by a nonradioactive
in situ hybridization method using cosmids of the short arm
of chromosome 16. The relative order of these cosmids is
known from family studies.I8 The in situ hybridization
showed that the breakpoint in the short arm of chromosome 16 in patients with an inversion 16 in ANLL M4eo is
located distal to cosmid 36 and proximal to cosmid 41. Our
patient with ANLL M5 had two translocated chromosomes
16, a t(8;16) and a t(11;16). Both breakpoints were cytoge-
RESULTS
Cytogenetics. Analysis of GTG-banded metaphases from
bone BM showed a 46,XX,-16,t(8;16)(pl l;p13), +der( 16)
[t(11;16)(q13;p13)] karyotype in 19 of 20 cells (Fig 1). The
phytohemagglutinin (PHA) stimulated blood culture showed
a normal karyotype.
GTG-bandinglchromosome painting. In standard GTGbanded metaphases, the translocated part of chromosome
16 (pl3-pter) is very difficult to detect on the short arm of
chromosome 8. Use of combined GTG-banding and fluorescent in situ hybridization with a chromosome 16 cosmid
library, however, showed this subtle chromosome rearrangement, ie, a positive signal on the translocated chromosome
8, which originated from chromosome 16. The t(11;16)
proved to be a nonreciprocal translocation (Fig 3, A and B).
Breaboint localization. Metaphase spreads of the patient were hybridized with cosmids 36,41, and 40. Cosmids
36 and 41 hybridized to both translocated chromosomes 16
(Fig 4), and cosmid 40 hybridized only to one of the
translocated chromosomes 16 and to the translocated
chromosome 8 (Fig 5). To show the specificity and the
distance between the cosmid probes 40 and 41, normal
metaphase spreads were hybridized (Fig 6). The t(8;16) and
the t( 11;16) appear to have different breakpoints on the
short arm of chromosome 16. The t(11;16) breakpoint could
be mapped distal to the cosmids 40, H17, cos-aIII, and
cos-S‘HVR I. This breakpoint must be very close to the
breekpolnI8 CY 18
11
CY 19
‘Y
GM2324
PK32-I
I
I
tlll $6)
lnvertlon 16
Fig 2. Map of the localization
of cosmids on the short arm of
chromosome 16 and localization
of the breakpoints in inversion
16, translocation 8;16, and translocation 11:16.
centrom*re
Cormldr
36
41
c------)
24
218
26
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DISTINCT LOCI ON SHORT ARM OF CHROMOSOME 16
1557
Fig 4. Nonradioactive in situ hybridization with pHUR195 centromere probe and cosmid 41. The cosmid probe hybridized to both
t(11;16) ("1 and t(8;16) (arrowhead).
Fig 5. Nonradioactive in situ hybridization with pHUR195 centromere probe and cosmid 40. The cosmid probe hybridized to
t(ll;l6) (*) and translocated chromosome 8.
Fig 3. (A) GTG-banded metaphase spread [arrow 1 indicates
t(8;16); arrow 2 indicates t(ll;l6)]. (6)Nonradioactive in situ hybridization (chromosomepainting)of the same metaphasewith a chromosome 16 specific cosmidbank.
>
Fig 6. Nonradioactive in situ hybridization with pHUR195 centromere probe and cosmids 40 (*) and 41 (arrowhead) on a normal
metaphase spread.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1558
WESSELS ET AL
netically localized to 16(p13). We wished to determine
whether these breakpoints were in the same breakpoint
interval (between cosmids 36 and 41) as in the inversion 16.
Using nonradioactive in situ hybridization with several of
the cosmid probes used by Dauwerse et
we showed that
the breakpoints in both translocations differ from the
localization of the breakpoint in the inversion 16.
Because no contiguous physical map of the entire 16(p13)
band exists, estimating the distance in megabases or kilobases (kb) between the breakpoints of the inversion 16 and
the translocation 8;16 is not possible. The breakpoint of the
translocation 11;16 in our patient must be very close to the
telomere of chromosome 16 because the a-globin cluster
has been mapped by P. Harris (personal communication)
within 100 kb of the telomere.
To our knowledge, no cellular oncogenes have yet been
mapped in the regions 8(pll) and 16(p13). Mitelman,"
suggested both breakpoints to be possible regions of pri-
mary chromosomal rearrangements characteristic of myeloid disorders. In the proliferation-specific/differentiationassociated chromosomal rearrangements model of Heim
and Mitelman,'" the translocation 8;16 appears to be one of
the few reciprocal translocations in which no oncogenes
have yet been reported. At present, the results show that in
inversion 16 and in translocation 8;16 different critical
genes in the short arm of chromosome 16 may be involved.
A larger series of patients with this same translocation
should confirm these results, but was not available.
ACKNOWLEDGMENT
We thank K. van der Ham for preparing the photomicrographs,
F.G.M. Snijdewint for preparing the cosmid map, M. Kielman for
the cosmids cos-a111and cos-5'HVR I, our colleagues for patience,
R. Kibbelaar and V. Smit for critical reading of earlier versions of
the manuscript, and M. Voorhans for encouragement.
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1991 77: 1555-1559
Two distinct loci on the short arm of chromosome 16 are involved in
myeloid leukemia
JW Wessels, P Mollevanger, JG Dauwerse, FH Cluitmans, MH Breuning and GC Beverstock
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