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Localization by Chromosome Microdissection of a Recurrent Breakpoint
Region on Chromosome 6 in Human B-Cell Lymphoma
By X.-Y. Guan, D. Horsman, H.E. Zhang, N.Z. Parsa, P.S. Meltzer, and J.M. Trent
Deletion of the long arm of chromosome 6 (6q) is one of
the most common
chromosomal alterations in human B-cell
lymphomas. Conventional cytogenetic banding
analysis and
loss-of-heterozygosity (LOH) studies have detected several
common regions of deletion ranging across the entire long
yet identified.
arm (6q1, with no defined recurrent breakpoint
We describe here a strategy combining chromosome microdissection and fluorescence insitu hybridization (MicroFISH) t o determine a minimal region of deletion along chromosome 6. Seven clinical cases and one cell line of follicular
lymphoma containing a t(14;18) and one case ofdiffuse
lymphoma, also with a t(14;18), were used for this study.
All ninecases had previously defined abnormalities of chromosome 6 determined by cytogenetic analysis. The results
of chromosome dissectionwere unexpected and in contrast
to thesuggestion of disparate breakpoints by conventional
chromosome banding. Specifically, Micro-FISH analysis provided evidence for a common breakpoint at 6 q l l in seven
of ninecases. After Micro-FISH analysis, all of the presumed
simple deletions of chromosome
6 were carefullyreanalyzed
and shown t o actually represent either nonreciprocal translocations (three cases), interstitial deletions (five cases), or
isochromosome (one case). The recurrent proximal breakpoint ( 6 q l l ) was detected in seven of nine cases, with the
minimal region of deletion encompassing 6 q l l t o6q21. By
analogy t o other tumor systems, the identificationof recurring breakpoints within 6 q l l may suggest that a gene(s)
important tothe
genesis or progression
of
follicular
lymphoma can be localized t o this band region.
This is a US government work. There are no restrictions on
its use.
H
phenotypez4 or the metastatic potential." Accordingly, identification of a recurrent site of chromosome breakage in follicular lymphoma may be of considerable importance in localizing a gene(s) involved
malignancies
in
with
chromosome 6 alterations.
In lymphomas and lymphoblasticleukemias, several commonlydeletedbandregions
along 6q have alreadybeen
reported, including(1)two regions of minimalmolecular
deletion (RMDs) termed RMDl (localized to 6q25-27) and
RMD2 (localized to 6q21-23) determined by loss of heterogeneity (LOH)," and (2) two common regions of deletion at
6q23.1-27 and 6q14-q21suggested in acute lymphoblastic
leukemia by fluorescence in situ hybridization (FISH) of
yeast artificial chromosome clones mapped to 6q.'.'' Unfortunately, despite these significant recent gains in our knowlof
edge of the breakpointson6q,
mostinvolveaseries
chromosomal bands. One significant technical problem that
continues to limitthis area ofresearchis our inability to
exactly characterize cytogenetic breakpoints
by conventional
G-banding analysis.
We have recently developed an approach (termed MicroFISH)*' that combineschromosome microdissectionand
FISHto rapidlycytologicallydefinethe
exact limits of
breakpoints involved in chromosome deletions. In this report, nine cases of B-cell lymphoma with identified chromosome 6 abnormalities have been studied by microdissection.
Using this method, all presumed chromosomal deletions involving 6q were shown to represent nonreciprocal translocations (three cases), interstitial deletions (five cases), or isochromosome (one case). In addition to the identification of
a RMD encompassing 6 q l 1 to 6q21, a recurrentproximal
breakpoint at 6ql1 has been identified.
UMANTUMORS HAVE beenshown
to progress
through a multistep process of genetic abnormalities,
which frequentlyinvolvesrecurring
chromosomalchange
mediating alterations in the expression of oncogenes or tumor suppressor genes.' A reciprocal chromosome translocation, t( 14; 18)(q32;q21), has been detected in approximately
85% of cases of follicular lymphoma studied by cytogenetic
molecular
or This
translocation
results
in the
juxtaposition of the oncogene, BCL-2, at 18q21 with the Ig
heavy-chain locus on 14q32.4 Cytogenetic studies have also
shown that deletion of the long arm of chromosome 6 (6q)
in all nonis oneof the most common chromosomal changes
Hodgkin's
including
follicular
lymphoma."',"
Deletion of 6q is thought to be associated with a clinically
aggressiveform of follicularmixed small-and large-cell
histologic type." Clinically, patients withan abnormal chromosome 6 also have a higher frequency of immunoblastic
l y m p h ~ m aFinally,
.~
nonrandom rearrangement of 6q is also
commonly
observed
in other hematopoietic
malignancies,lz,13including acute lymphoblastic leukemia,'"l5 and in
many solid tumors, includingmalignant melanoma,l"'Xovarian carcinoma,'"'' and breast carcinoma."^" Evidencefor
the biological importance of chromosome 6alterations in
malignancy has been provided by the introduction into tumor
cells of a normal human chromosome6 after microcell transfer and the resulting suppression
of either the tumorigenic
From the Laboratory of Cancer Genetics, National Center for
Human Genome Research, National Institutes of Health, Bethesda,
MD; and the British Columbia Cancer Agency, Division of Labomtory Medicine, Vancouver, Canada.
Submitted December 18, 1995; accepted April 8, 1996.
Address reprint requests to J.M. Trent, PhD, NCHGWNIH, Bldg
49, Room 4A22, 49 Convent Dr. MSC 4470, Bethesdu, MD 208924470.
The publication costs of this article were defrayedin part by puge
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
This is a US government work. There are no restrictions on its use.
0006-4971/96/8804-0031$0.00/0
1418
MATERIALS AND METHODS
Cytogenetic unuIysis. Cytogeneticanalysis was performed on
blood, bone marrow, and lymph node specimens as previously described.' Metaphases were G-banded, and abnormal copies of chroSU-DHL-4,2"
mosome 6 fromthefollicularlymphomacellline,
andeight B-cell lymphoma biopsy specimenswereanalyzed
by
chromosome microdissection. Tissue specimens were obtained from
biopsies of peripheral blood (9322803. bone marrow (91C037), and
Blood, Vol 88, No 4 (August 15), 1996: pp 1418-1422
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AKPOINT
RECURRENT
IN B-CELL LYMPHOMA
1419
Table 1. Cytogenetic Alterations From Eight Patients With B-Cell Lymphoma
Patient No.
Clone
47,XY,add(4)(q2?),de1(6~~q15~,der(8~t~8;17~(pll;qll~,add~10~~q22~,
der(6)t(5;6)(~13;qll)
+13,t(14;18)(q32;q21),-17,+mar(?Y)[61/46,XY[ll
1: 80-88,XXW,+X,(del)(l)(qll),der~l~~q13~,-6,de1~6~~q21q27~,de1~1l~~q23~,t~14;18~
de1(6)t(6;16)(qll;pll)
Clone
91C313
(q32;q21),inc[cp151/46,XY[31
46,XY,add~l~~p36~,de1~6~~q15~,t~14;18~~q32;q21~~151/46,XY~21
46,XX,de1(6)(q13q22~,t~14;18~~q32;q21~[101/46,XX141
92C147
92C326
92C390
93C280
6q Interpretation by Micro-FISH
Original Karyotype Interpretation
91C0371
1:
Clone
1:
Clone
1:
Clone
1:
de1(6)(qllq21)/(q22q25)
del(6Nqllq22)
50,X,del(X)(q26),t(2;19)(19;4)(q23;q13p13;q22~,+de1~3~~pl4p23~,de1~6~~ql3q23~, deI(6Hqllq22)
der~6~t~6;14~(q21;qll~,add~7~~q36~der~7~t~8;?;7~~7;13~~qll;?;p22~~q36;q13~.-14,+add~l5~
(q2l),is0~18)(q10),+add(18)~q23)~2[61
1: Clone 44-45,Y,add(X)(p22),de1(2)(?ql3ql4),del~3~~pl2p25~,?dic~4;l7~~pl6;pll~,der~5~t~l;5~
del(6)(q12q22)
(q21;p14),add(6)(q13),add(8)(q22),add(9)(pl3~,add~lO~~q24~,add~ll~~q25~,del~l3~~ql2q32~,
der(14)t(12;14;18)(q24.l;q32;q21),der~18~t~12;14;18~~q24.l;q32;q21~~cp51
94C016
94C180
previouslySU-DHL-4
Clone 1: 95-100,XXXX,+Xx2,add(l)(pl1)x2,+3,-4x2+de1~6~~q21~x2,+7,de1~7~~q32q36~,+8x2, der(6)t(6;8)(p12;qll)
+add(8)(pll),+ll,+12x2,+t(14;18)(q32;q21)x2,-15x2,-16,-17,-18,+20,+21,+2mars~cp81/
46,XX[121
Clone 1: 48,X,-Y,del(l)(?p35p36),t(3;22)~q27;qll),+5,-6,del~6~~ql3q25~x2,+7,t~l4;l8~~q32;q2l~
deI(6Mqllq24)
[151/46,XY[61
Karyotype
iso(6)(qll)
lymph node (9lC3 13, 92C147, 92C326, 92C390, 94COI 6.and
94CI80). Mitotic cells were dropped onto clean cover slips (22 X
60 mm) and stored at37°C for 2 to 3 days. Standard G-banding
with trypsin-Giemsa was performed before microdissection.
Microdissection and topoisomerase I treatment. The procedure
for chromosome microdissection was performed essentially as described previously.” Briefly, two to five copies of target rearranged
chromosome 6 were dissected with glass microneedles controlled
by a Narishige micromanipulator (Narishige USAInc. Sea Cliff,
NY) attached to an inverted microscope. The dissected chromosome
fragments were transferred to a S-pL collection drop (containing 40
mmol/L Tris hydrochloride, pH7.5. 20 mmol/L MgCl?, SO mmoll
L NaCI, 200 pmol/L of each dNTP, 0.1 U topoisomerase 1. and S
pmol universal primer
[UN
I ] CCGACTCGAGNNNNNNATGTGG). After the desired number of dissected DNA fragments was
collected, the collection drop was covered with a drop of mineral
oil, incubated at 37°C for 30 minutes, and denatured at 96°C for I O
minutes.
Amplification of dissected DNA. An initial eight cycles of polymerase chain reaction ([PCR] denaturation at94°C for I minute,
annealing at 30°Cfor 2 minutes, and extension at 37°C for 2 minutes)
were performed by adding approximately 0.3 U T7 DNA polymerase
(Sequenase version 2.0: USBlAmersham Life Sciences, Inc. Arlington Heights, IL) at each cycle (Sequenase 13 U/pL was diluted 1:8
in enzyme dilution buffer [US Biochemical]. and 0.2 pL was added
to 5 pL reaction mixture). Following this preamplification step. a
conventional PCR reaction catalyzed by Taq DNA polymerase was
performed in the same tube. PCR reaction mix (SO pL) was then
added ( I O mmol/L Tris hydrochloride, pH 8.4. 2 mmol/L MgCI?.
SO mmol/L KCI, 0. I mg/mL gelatin. 200 pmol/L of each dNTP, SO
pmol UN1 primer. and 2 U Taq DNA polymerase [Perkin-Elmer/
Cetus]). The reaction was heated to 95°C for 3 minutes followed by
35 cycles of 1 minute at 94°C. I minute at 56°C. 2 minutes at 72°C
and a S-minute final extension at 72°C.
FISH. Amplified microdissected DNA (2 pL) was labeledwith
Biotin-16-dUTP (Boehringer Mannheim Biochemical, Indianapolis,
IN) in a secondary PCR reaction identical to that already described
except for addition of 20 pmol/L Biotin-16-dUTP. The reaction was
continued for 15 to 20 cycles of 1 minute at 94°C. 1 minute at 56°C
and 2 minutes at 72°C. with a S-minute final extension at 72°C. The
a Centricon 100 (Amicon.
PCR products werethenpurifiedwith
Beverly, MA) filter and used for FISH. Hybridization of the FISH
probes followed our procedure described previously,’”whichis
based on the procedure of Pinkel et al.’*
RESULTS
9 1C037
92C
936280
147
926390
94CO 16
94C 180
Fig 1. Examples of rearranged chromosome 6 in B-cell lymphomas. Partial karyotypesof normal (left)and abnormal (right) chromosome 6 for 7 cases. Specifickaryotypic information on all alterations
is provided in Table 1.
Cytogenetic ana!\psis. Cytogenetic analysis was
performed on all cases. Table 1 summarizes the karyotype alterations interpreted by conventional G-banding analysis of the
clinical samples. Six of eight primary tumor cases contained
an abnormal chromosome 6 with an apparent terminal deletion of 6q (Fig l ) . The breakpoints determined by banding
analysis were between bands 6q15 and 6q22 (Table 1). The
remaining two primarytumor cases eachcontained rearranged chromosome 6 with undefined addition of material
on 6q at 6q 1 1 and 6q 13. The lymphoma cell line, SU-DHL4,contained an isochromosome of the short arm of chromosome 6. Examples of the abnormal copies of chromosome
6 chosen for microdissection are illustrated in Fig 1.
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1420
GUAN ET AL
Fig 2. Detection of a complex interstitial deletion involving chromosome 6 in a primary lymphoma (92C1471.(AI Representative normal Gbanded lymphocyte metaphase. (61Metaphase identical to (AI after FISH with e probe generated from the microdissected DNA recognizing
a complex interstitlal deletion [der(6)del(qllq21)deI(6)(q22q2511.
Micro-FZSH. To identify the chromosomal composition
of additional material on 6q and to precisely determine the
breakpoints of6q deletions, microdissection of the entire
abnormal chromosome 6 was performed in all nine cases.
Briefly, two to five copies of the entire rearranged chromosome 6 in each case were microdissected, andDNA was
directly amplified byPCR and labeled with Biotin-16-dUTP
in a secondary PCR reaction. The labeled probe was then
used for FISH to normal peripheral blood lymphocyte metaphases. Sequential G-banding and FISH were performed using the probes generated from the microdissected chromosome 6samples, allowing definition of breakpoints. The
results were unexpected compared with those suggested by
G-banding analysis. The chromosomalcompositionand
Fig 3. Detection of a cryptic chromosome translocation of chromosome 6 in case 91C037.(A) Representative normal G-banded lymphocyte
metaphase. (B)Same metaphase hybridized with a micro-FISH probe recognizinga nonreciprocal translocation [der(6)t(5;6)(pl3;qll)].
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1421
RECURRENTBREAKPOINT IN B-CELL LYMPHOMA
consistent region of minimal deletion observed in u I I nine
cases encompassed 6q I I to 6q2 1 .
DISCUSSION
T
’ T
‘ T
T
T
.l
Fig 4. Minimal deletion map and distribution of breakpoints on
chromosome 6 from all 9 casesofB-cell lymphoma. Deletions of
chromosome 6 are indicated by vertical solid lines, with breakpoints
indicated by horizontal solid lines. The RMD identified encompassed
6q12 to 6q21.
chromosome 6 breakpoints for all cases are summarized in
Table I . In contrast to the G-banding analysis. none of the
rearranged chromosome 6 samples were shown t o be terminal deletions. Instead. in tive of eight cases (WC 147.
02C326, 92C390. 93C280. and 94C 180) interstitial deletion
of 6q was observed. Interestingly. the proximal breakpoint
on 6q from four of five cases was localized at 6q I I . In the
remaining case, the proximal breakpoint was defined at6q 12.
The distal breakpoints involving chromosome 6 interstitial
deletions were variable (Table I ). An example of a complex
interstitial deletion involving 6q that resulted in the deletion
of two separate band regions (6q I I-q2 I and 6q22-q2S) was
detected in case 92C147 (Fig 2).
In three of nine cases analyzed (91C037, 91C313, and
94C0 16) the rearranged chromosome 6 was demonstrated to
represent a nonreciprocal translocation (Table I ) . In two of
three cases, the breakpoint on 6q was localized to 6q I I . In
the remaining case. the breakpoint occurred in the short arm
( 6 ~ 1 2 )The
. chromosomes participating in the nonreciprocal
translocation were derived from three different chromosomes: 5. 8. and 16 (Table I ). Figure 3 shows an example
of a nonreciprocal translocation from case 91C037 demonstrating a t(S:6) translocation [der(6)t(S:6)(pl3:ql l ) ] . In the
remaining case (SU-DHL-4) the rearranged chromosome 6
was demonstrated by micro-FISH to represent an isochromosome of 6p. with the breakpoint again localized to 6q I 1 .
Figure 4 summarizes the deletion map and distribution of
breakpoints for all nine cases with 6q rearrangements. The
Loss of genetic material on the long arm of chromosome
6 is commonly detected in human B-cell lymphonla by both
cytogenetic and LOH analyses. These and other studies
strongly support the notion that a tumor suppressor gene(s)
involved in lymphoma biology is localized to 6q. To date,
;I wide distribution of breakpoints along 6q havebeen reported in non-Hodgkin‘s lymphomas (including follicular
lymphomas). SeverA common regions of deletion have been
reported in non-Hodgkin’s lymphomas. including 6q2 14423
and 6q25q27.” 6q2 I -6q25.’.” 6q 14-2 1, and 6q73-27.‘ Although the banding quality obtained from cases of follicular
lymphoma is often acceptable. it remains difticult to interpret
thebandingpattern subsequent to a deletion. This may in
Inrge part reflect therepetitive nondistinctive banding pattern
of 6q. The fact that other investigators have not appreciated
the proximal nature of these deletions supports this notion.
As a consequence. no recurrent breakpoint information has
been available to facilitate positional cloning studies in this
disease.
In this study. two important and novel findings have been
made regarding chromosome 6 rearrangements in follicular
lymphoma. First, it was striking thatall cases of6q rearrangement defined by cytogenetic analysis as terminal deletions were. in fact. demonstrated to be either interstitial deletions or nonreciprocal translocations. The extent of the
deletion of the long arm of chromosome 6 among each case
varied consitlerubly in this study. Four cases lost the entire
6q. with the remaining tive cases showing partial loss of the
long arm of chromosome 6 (Fig 4). The common RMD was
determined to be 6q I I -6q2 I . Comparison of our results to
those previously derived from cytogenetic and LOH studies
suggests that the RMD in this disease may be more proximal
than previously recognized. This results from previous chromosomal breakpoint assignments made on the basis of Gbanding. and the relative lack of tested proximal
polymorphic markers along 6q.
Second. we have
identified
a recurrent proximal
breakpoint on 6q in human follicular lymphomas at 6ql 1.
In this study. seven of nine cases demonstrated a rearranged
chromosome 6 with the proximal breakpoint at 6ql1. Four
cases of interstitial deletion had the proximal breakpoint at
6q I I . whereas in two cases of nonreciprocal translocations
and one case of isochromosome 6p. the breakpoint occurred
in 6ql I . This raises the possibility that therecurrent breakage
at 6ql I could represent either a primary chromosomal
change interrupting a gene of importance to the biology of
follicular lymphoma, or merely a region of “fragility” that
is broken frequently because of some structurally unique
feature. To clarify this question. studies are currently under
way in our laboratory using region-specific microclone libraries to facilitate positional cloning of this breakpoint.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1996 88: 1418-1422
Localization by chromosome microdissection of a recurrent
breakpoint region on chromosome 6 in human B-cell lymphoma
XY Guan, D Horsman, HE Zhang, NZ Parsa, PS Meltzer and JM Trent
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