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Detection of myc Translocations in Lymphoma Cells by Fluorescence In Situ
Hybridization With Yeast Artificial Chromosomes
By Maria Luisa V e r o n e s e , Masataka O h t a , Janet Finan, Peter C. Nowell, a n d Carlo M. Croce
Translocations involving chromosome 8 at band q24 and one
of the Ig loci on chromosomes 14q32,22qll, and 2 p l l are
the hallmark ofBurkitt’s lymphoma (BL). It has been previously
observed
that
the
exact localization of the
breakpoints at chromosome 8q24 can varysignificantlyfrom
patient to patient, scattering over a distance of more than
300 kb upstream of c-myc and about 300 kb downstream of
c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myctranslocations,
we
screened a yeast artificial chromosome (YAC) library from
normal human lymphocytes by colony hybridization, using
three markers surrounding the c-myc gene as probes. We
obtained 10 YAC clones ranging in size between 500 and
200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different
breakpoints at 8q24. Our results show that the YAC clones
detected translocations scattered along approximately 200
kb in both metaphase chromosomes and interphase nuclei.
The sensitivity, rapidity, and feasibility in nondividing cells
render FISH an important diagnostic tool. Furthermore, the
use of large DNA fragments such as YACs greatly simplifies
the detection of translocations with widely scattered
breakpoints such as these seen in BL.
0 1995 by The American Society of Hematology.
T
good mitotic index and good quality of metaphase spreads.
The scattering of breakpoints and therefore the necessity of
multiple probes and sequence-specific primers render Southe m blot analysis and PCR too time-consuming for routine
diagnosis. Fluorescence in situ hybridization (FISH) can be
used to overcome these 1imitati0ns.l~ FISHis a rapid technique, its specificity and sensitivity are very high, its detection of chromosomal abnormalities is independentofthe
cycling status of the cells,I5 and it permits a direct correlation
of cytogenetic findings with cell morphology.“ Moreover,
theability touse differentlabeling
systems, suchas in
multicolor FISH,I7 and different types of probes render it
highly flexible. Indeed, FISH is not limited by probe size or
probe type, and probes that identify a specific chromosomal
structure,Ix probes that hybridize
to multiple chromosomal
sequences,” and probes that hybridize to unique sequences
may be used alone or in combinations to better define complex chromosomal translocations in diagnosis and in basic
research. Because FISH is not hindered by the presence of
repeat sequences, large genomic probes, such as yeast artificial chromosomes (YACs) capable of covering a large region, can be used to overcome the need for multiple probes.
In thisstudy, we present data from two-colorFISHon
severalBurkitt’scelllines
andfreshtumorsamplesfrom
patients with BL. Two YAC probes,I2 and P72, were usedto
analyze both metaphase chromosomes and interphase nuclei.
Results indicate that these two YACs effectively detect the
different breakpoints on chromosome 8 in both metaphase
chromosomes and interphase nuclei.
RANSLOCATIONS involving thec - m y locus on chromosome 8, although characteristic of Burkitt’s
lymphoma (BL),’ have also been documented inacquired
immunodeficiency syndrome(AIDS) lymphomas,*diffuse
large-cell lymphomas,.’ and acute T-cell leukemias4 These
translocations juxtapose the c-myc oncogene located at 8q24
to either the Ig loci for the heavy chain or the K and X light
chains located at 14q32, 2pl1, and 22ql1, respectively, or
“
all of
to the a/6 locusofthe T-cell r e ~ e p t o r . ~Although
c-myc, the
these translocations result in the deregulation of
location of thebreakpointsonchromosome
8 can vary
greatly from patient to patient. In the most frequent translocation, t(8; 14), the breakpoints on chromosome 8 are clustered within or near the c - m y locus as in the sporadic types
of BLs7 or, alternatively,may
bedispersedoverabout
300 kb upstreamof thegene,as in theendemic African
cases.”’ Studies in AIDS-related BL, although relatively limited,indicatethat
thebreakpointsare scatteredin similar
fashion both 5’ and 3’ to c-myc.“’.” In the variant translocations t(8;22) and t(2;8), the breakpoints are scattered over
a distance of about 300 kb downstream of c-myc.”
Because they provide a cytogenetic and molecularmarker
for diagnosis and follow-up of the disease, these translocations have been analyzed cytogenetically, by Southern blot
analysis, and by the polymerase chainreaction(PCR).”
However, although cytogenetic analysis allows detection of
all of these translocations, the technique is
dependent on a
From the Jefferson Cancer Institute, Jrfferson Medical College,
Philadelphia, PA: and the Department of Pathology and Laboratoy
Medicine, Universin. of Pennsylvania, Philadelphia, PA.
Submitted October IO, 1994; accepted November 8, 1994.
Supported by anOutstanding Investigator Awards (CA 39860 and
42232) fromthe National Cancer Institute (C.M.C. and P.C.N.) and
an AIDS fellowship( D M . 4.16.92, No. 26O/O/SAP 7.2)from lstituto
Superiore di Sanita, Italy (M.L.V.).
Address reprint requests to Maria Luisu Veronese, MD, Jefferson
Cancer Institute, Jefferson Medical College, BLBS, Room 1032, 233
S 10th St, Philadelphia, PA 19107.
The publicationcosts 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 1995 by The American Society of Hematology.
0006-4971/95/8508-0007$3.00/0
2132
MATERIALS AND METHODS
Cell lines andpatients. The BL cell lines Daudi,P3HR1, AG876,
and EW36 (kindly provided by
I. Magrath,NationalInstitutes of
Health, Bethesda, MD); Manca and BL2 were
used in this study.
All cell lines carry translocations involving chromosome 8 at band
q24. Each of the five patient samples studiedhad a t(8;14) translocation. Four of the patients were diagnosed with sporadic BL (sBL)
andone was diagnosed with large-cellimmunoblasticlymphoma
(LCIL; see Table 2).
Isolation and characterization of YAC clones. A YAC llbrdry
established from normal human lymphocytes’” was screened by colony hybridization, as previously described.” with probes pRyc7.4,”
of isolated YAC clones
pPAl.3SB,” and pD4AHH1.6.” The size
was determinedby pulse-field gel electrophoresis (PFGE) and probes
8q267,” pEW36-9H2.0,Xp380-8AI,S,” p380j9 0.8~s.” ~ E w 3 6 - 7 D . ~
Blood, Vol 85,No 8 (April 15).
1995:
pp 2132-2138
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DETECTION OF
MYC
2133
TRANSLOCATIONS BY FISH
G4
E2
t
I
I2
I
P72
I
I
Mance
-IT 1""
BU
II
a
3'
5'
and H4.lZ6were used by Southern blot analysis to map their location
within the 8q24 breakpoint region.
FISH. Metaphase chromosomes from cell lines were prepared
according to standard techniques." Patient samples were cultured in
Chang in situ Medium (Irvine Scientific, Santa Ana, CA) at 37°C
with 5% CO2for 24 hours and metaphase spreads were then prepared
in the same manner as cell lines. Slides were pretreated with RNase
A ( J 00 pgimL; Boehringer Mannheim, Mannheim, Germany) in 2X
SSC (pH 7.0) for 60 minutes at 37"C, followed by two washes in
2 x SSC and sequential dehydration in 70%, 85% and 100% ethanol.
Slides were then denatured in 70% formamide, 2X SSC at 70°C for
2 minutes and dehydrated. YAC clone DNAwas labeled by nick
translation with biotin-14dATP (BRL, Gaithersburg, MD), coprecipitated with I O to 15 pg of unlabeled human Cot-l DNA (BRL), and
resuspended in 30 p L of hybridization solution (50% formamide,
10%dextran sulphate). The probe was denaturated, allowed to preanneal at 37°C for 1 hour, and combined with the denaturated digoxigenin-labeled a satellite probe D14Zl/D22ZI (Oncor, Gaithersburg,
MD) or with the Te114q(14q32.3-qter) probe (Oncor) immediately
before adding the mixture to the slides. After overnight hybridization
and washes in 50% formamide 2x SSC at 45°Cand 2X SSC at
37°C. the digoxigenin- and biotin-labeled probes were detected immunologically using fluorescein isothiocyanate conjugated to avidin
(FITC-avidin DCS; Vector Laboratories, Burlingame, CA) and rhodamine-conjugated antidigoxigenin antibodies (Oncor). The chromodihysomes were counterstained with 4,6-diaminido-2-phenylindole
drochloride (DAPI) and visualized with a triple band pass filter from
Chromatech (Brattleboro, VT).
RESULTS
Ten YAC clones were isolated from the YAC library after
screening with probes pRyc7.4, pPA1.3SB, andpD4AHH1.6
(Fig 1). PFGE indicated sizes ranging between 200 and 500
kb. All clones were analyzed by Southern blotting using
the probes 8q267, pEW36-9H2.0, p380-8A1.8, p380j9 0.8ss,
pEW36-7D, and H4.1, in addition to the probes used for the
screening of the library to define overlaps and to map the
clones with respect to known breakpoints around the c-myc
locus. The clones were further screened for chimerism by
FISH on normalhuman lymphocyte chromosomes. Two
nonchimeric clones, P72 and I2 (which gave a strong signal
on both chromatids of chromosome 8 only), were used to
detect the chromosomal breakpoints in the Burkitt's cell lines
and patient samples.
Four YAC clones forming a contig spanning the c-myc
locus are depicted in Fig 1, which shows their relative sizes
and position with respect to c-myc. Clones E2 and G4 were
not used for FISH on BL cell lines or fresh tumor samples,
because E2 showed cross-hybridization with the centromere
of D group chromosomes and G4 was shown to be chimeric.
12 contains an insert of approximately 200 kb that includes
the c-myc gene and the pPAl.3SB marker. The 8q267
marker, located approximately 60 kb downstream of c-myc,
is not present in the insert that we were then able to position
with respect to c-myc as shown inFig 1 . P72 is approximately 500 kb in size and contains the p380-8A1.8, p380j9
0.8ss, pEW36-7D, and H4.1 markers.
To determine the effectiveness of our YAC clones in detecting different breakpoints on chromosome 8 both in metaphase chromosomes and interphase nuclei, we performed
two-color FISH analysis on six BL cell lines, five of them
carrying the t(8; 14) translocation and one the t(8;22) translocation (Table 1). We also examined tumor samples obtained
from five patients whose karyotype had shown a t(8; 14)
Table 1. Location of Chromosomal Breakpoint in BL Cell Lines and
FISH Results With YAC Clones P72 and 12
Cell Line
BL2
Daudi
AG876
P3HRI
EW36
Translocation
t(8;22)
t(8; 14)
t(8: 14)
t(8; 14)
t(8; 14)
Breakpoint on
Chromosome 8'
FISH Signals With 12
and P72
20 kb 3'
170-190 kb 5'
Unknown
170-190 kb 5'
170-190 kb 5'
N8, der (81, der (22)
* Breakpoint positions are given with
N8, der
N8, der
N8, der
N8, der
(8). der
(81, der
(8). der
(E),der
respect to c-myc.
(14)
(14)
(14)
(14)
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2134
VERONESE
ET AL
Table 2. Diagnosis and Cytogenetic Findings in Five Patients
Patient
No.
Sex/Age
1
MI10
2
F125
3
4
5
M13
F145
(v)
M16
Source
Specimen
Diagnosis
SBL
SBL
SBL
LClL
SBL
Peritoneal
effusion
Bone marrow
Bone marrow
Lymph
node
Bone marrow
46,XY,add(l)(q?),t(8;14)(q24;q32)
46,XX,del(3)(pl2p24),t(8;14)(q24;q32)
46,XY,t(8; 14)(q24;q32)
49,XX,-2,+3,de1(6)(q21,q23),+7t(8;14)(q24;q32),add(9)(p22-24),+13,i(18q),+mar
46,XY,t(8;14)(q24,q32)
BL2, Daudi, and AG876. The breakpoints on chromosome
translocation (Table 2). Biotin-(green) labeled clones p72
and I2 were combined with digoxigenin-(red) labeled centro- 8 of the BL2 and Daudi cell lines have been well charactermeric probe D14Zl/D22Zl or Tell4q probe. The D14Z1/
ized. The BL2 breakpoiit is located 10 kb downstream of
D22Z1 probe hybridizes with the centromeres of chromoc-myc2 and the Daudi breakpointis 170 to 190 kb
upstream
somes 14 and 22, whereas the Te114q probe detects only
the
of c-myc? The exact location
of the breakpoint in the AG876
telomere of chromosome 1%. Using this approach, we were
cell line is not yet defined. When applied on the BL2 cell
line (Fig 3A and B) carrying the t(8;22) translocation,
able to identlfy unequivocally the partner chromosome in
I2
metaphase spreads, whereas, in interphase nuclei, the presand P72 hybridized to normal chromosome 8 (green dots)
ence of the translocation was demonstrated by the presence and splitsignalsonboththeder(8)andder(22).
On the
of three green signals (the biotin-labeled YAC),ofone
which
Daudi and AG876 cell l i e s (Fig 3C and D) carrying the
colocalizeswithoneredsignal(thedigoxigenin-labeled
t(8; 14) translocation, both clones hybridized to the normal
chromosome 8 and to both the der(8) and der(l4) chromoD14ZUD22Zl or Tell4q probes; Fig 2). Figure 3 shows
representative results of FISH analysis on three cell lines:
somes. The breakpoint in the AG876 cell line
is shown here
to be located within the region covered by our YACs.
I2
and P72 also detect the t(8; 14) translocationin the P3HRI
andEW36 cell lines with a breakpoint at 170 to 190 kb
A
upstream of c-myc.* Analysis of the Manca cell line,
in which
the breakpoint occurs within thec-myc gene:'
showed that
both YAC clones effectively detect the t(8; 14) translocation.
FISH analysis on patients samples was performed using
the two YACclonestogetherwiththe
Tell4q probe.
Breakpoints in tumor samples have not been characterized.
Figure4A,B,and
D showsrepresentativemetaphases,
whereas an interphase is depicted in FigF- 4C.cells, including both metaphases and interphases, were examined
for each
patient. In fourpatients(no. 1 through4),themajority
of
8
14
cells,from30to35
(60% to70%),showedthreesignals
and colocalization of the YAC clones with theTell4q probe,
indicating
that
the
YAC
clones
effectively
detect
the
breakpoint on chromosome 8. In 5 to 10 cells (10% to 20%),
only two green signals were visible, one of which colocalized
B
with the Tell4q probe, indicating the presence of the der(8)
chromosome.Thenumberofnormalcells,withonly
two
green andtwo red signals without colocalization, ranged from
5 to 15 cells (10%to 30%). In patient no. 5, only interphases
wereanalyzedand two greensignalswerevisible,oneof
which colocalized with the Tell4q probein75%ofcells,
again demonstrating the presence of the der(8) chromosome.
uu
DISCUSSION
8
8q-
14 149+
Rearrangementsinvolvingthe
c-myc oncogene are the
hallmark Of BL*
BL3with the
transFig 2. Schematic illustrationof the detectionof the t(8;14) translolocations and C-mYC activation, represents the mostfreSUent
cation by dualcolor FISH. Green dots represent YAC clones 12 and
P72 on normal chromosome
8; red dots mark the telomere on normallymphoma developing in AIDS patients? In all translocachromosome14.Partialkaryotypesareshownon
the left and the
tions, overexpression of c-myc results from its j u w s i t i o n
corresponding interphase
nudei on the right. (A) Normal cell without
to Ig or T-cell receptor enhancers?' c-myc is overexpressed
t(8;14) translocation.(B) Detection of the t(8;14) translocation. Three
-ween sianals
- are visible, one of which colocalizes with a chromo- even when the breakpoints Occur more than 150 kb from the
some
14-specific
signal).
probe
(red
gene:3 indicating
enhancers
the
that
active
are
over
large
"
"
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DETECTION OF MYC TRANSLOCATIONS BY FISH
2135
Fig 3. Representative results
of FISHanalysis with YACs 12
and P72 on BL cell lines. (A) BL2
metaphase. (B) DAPl image of
same metaphase as in (A). (C)
Daudi metaphase. (D) AG876
metaphase. Long arrows mark
the position of normal chromosome 8,arrowheads indicatethe
der(8). and short open arrows indicate the der(22) (A and B) or
the der(l4) (C and D). The red
signal marks the centromeresof
chromsomes 22 and 14 (A and C)
or the telomere of chromosome
14 (D). In (D), the der(8) is labeled
by both the YAC clones (green)
and the Tell4q probe (red).
distances and allowing greatflexibilityin the location of
breakpoints. Indeed, this phenomenon is well
documented8.'.'' and has so far generally limited the use of molecular diagnostic methods in BL.
Using nonchimeric YAC clones containing large DNA
fragment surrounding the c-myc locus, we examined the efficacy of FISH analysis on metaphase spreads and interphase
nuclei in the detection of the t(8; 14) translocation characteristic of BL. Six BL cell lines with known breakpoints at
8q24 and five patient samples with unknownbreakpoint were
used as a model in this study.
Cytogenetic analysis, Southern blotting, andPCRhave
been widely used for the detection of rearrangements and
translocations. However,theneed
of good quality metaphases and a good mitotic index constitute major limitations
in cytogenetic analysis. Southern blot analysis can detect a
clone of cells representing as little as 1% of the total cell
population and it is suitable for observing patients serially.
However, only one probe can be used at a time and only
some rearrangements can be detected with one probe, making it cumbersome for routine diagnosis and follow-up. Because of its great sensitivity, PCR could provide a valuable
means of detecting chromosome translocations and localizing breakpoints to specific genetic regions. PCR has been
used in the detection of the bcr-ab1 transcripts in chronic
myeloid leukemia and adult acute lymphoblastic leukemia
carrying the t(9;22) transl~cation.~'
It has also been successfully used inthe detection of bcl-2 rearrangements in follicular lymphomas with the t(14; 18) transl~cation.~'
However,
it is limited to rearrangements of whichthe sequence is
known and that are clustered in a well-defined region,again
making this method impractical in the case of BL.
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VERONESE
*
A
Since its development, FISH has had great impact on diagnosis and basic research. FISH is a quick and simple method
that is highly specific and independent on the cycling status
of the cells. In addition, the feasibility of direct correlation
between cytogenetic and cytologic/morphologic features
allow accurate diagnosis even in equivocal cases.32The possibility of unequivocally identifying chromosomes with
chromosome-specificprobes simplifies the recognition of numerical and other cytogenetic abnormalities. In addition, the
use of combinations of differently labeled probes allows
high resolution ordering and mapping of DNA sequences.
Because FISH is not limited by probe size or by the presence
of repetitive sequences, large genomic fragments such as
YACs can be used in the analysis of chromosomal abnormalities. The advantage of YAC clones, covering several hundred kilobases within a chromosome region of interest, has
been shown in the case of acute myelogeneous leukemia for
the detection of the t(8;21) translocation, bcr-abl fusion in
chronic myeloid leukemia,33 and translocations involving
i
ET AL
Fig4.Representative
results
ofFISHanalysis
with YACs 12
and P72 on patient samples. (A)
Metaphase from patient no. 1.
(B) DAPl image ofsame metaphaseas in (A). (C) Interphase
from patient no.1.
(D)Metaphase from patient no. 4. Long
arrows mark the position of normal chromosome 8, arrowheads
indicate the derl8). and short
open arrows indicate the
der(l4). The red signal marks the
telomere of chromosome 14.
chromosome 11 at q23 band.34YAC clones were also used
to identify the partner sites of 14q32 translocations in B-cell
malignancies, even in cases not identified by cytogenetic
analysis.35
These characteristics make FISH ideally suited for the
analysis of the translocations in BL. Indeed, a pool of bacteriophage clones has been used to detect the breakpoint of
Burkitt's cell lines.36However, more than 30 phage clones
measuring 20 kb each would be necessary to cover the approximately 600-kb region involved in the rearrangements
and their combined use is too time consuming to be performed routinely. For this reason, larger probes such as
YACs covering a large region surrounding the breakpoints
represent a great advantage.
In this study, we have used two YAC clones spanning
approximately 700 kb surrounding the c-myc locus to detect
the different breakpoints of six Burkitt's cell lines. Twocolor FISH analysis shows that the clones effectively detect
the breakpoints. We also presented data from tumor samples.
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DETECTION OF
MYC
2137
TRANSLOCATIONS BY FISH
In four of them, the hybridization experiments showed both
the der(8) and der(l4) of the t(8; 14) translocation. In patient
no. 5 , the presence of only two green signals suggests that
the breakpoint is in the 3‘ region of c-myc, outside the region
covered by our clones. This problem can be easily overcome
by the isolation of a YAC clone extending further 3’ than
the contig presented in this work. Although studies including
a larger number of patients are necessary, our ability to
detect the presence of the translocations using the approach
described here indicated that it represents a sensitive and
specific tool in confirming a histopathologic diagnosis of BL
in cases in which classical cytogenetic analysis is not possible. In addition, three-color FISH could be used in the same
manner described in this study to differentiate between the
t(8; 14) and variant t(8;22) and t(2;8) translocations. The
ability to detect a tumor-specific genetic marker may have
significant impact not only in diagnosis but dso in the therapy and in follow-up of disease. The same approach could
also prove particularly relevant in other types of non-Hodgkin’s lymphoma that show the same variability in breakpoint
locations.3
1
ACKNOWLEDGMENT
We thank Florencia Bullrich and Massimo Negrini (Jefferson Cancer Institute, Jefferson Medical College, Philadelphia, PA) for their
helpful comments and assistance in the preparation of the manuscript. The cooperation of the LeukemiaLymphoma Study Group of
the University of Pennsylvania Cancer Center and of the Children‘s
Hospital of Philadelphia is also gratefully acknowledged.
REFERENCES
1. Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo R,
Croce CM: Humm’c-myc onc gene is located on the region of
chromosome 8 that is translocated in Burkitt lymphoma cells. Proc
Natl Acad Sci USA 79:7824, 1982
2. Levine AM: AIDS-related malignancies: The emerging epidemic. J Natl Cancer Inst 85:1382, 1993
3. Ladanyi M, Offit K, Jhanwar SC, Filippa DA, Chaganti RSK
Myc rearrangement and translocations involving band 8q24 in diffuse
large cell lymphomas. Blood 77:1057, 1991
4. Erikson J, Finger L, Sun L, ar-Rushdi A, Nishikura K, Minowada T, Finan J, Emanuel BS, Nowell PC, Croce CM: Deregulation
of c-myc by translocation of the T-cell receptor in T-cell leukemias.
Science 232:884, 1986
5. Erikson J, Nishikura K, ar-Rushdi A, Finan J, Emanuel B,
Lenoir G, Nowell PC, Croce CM: Translocation of an immunoglobulin K locus to a region 3’ of an unrearranged c-myc oncogene enhances c-myc transcription. Proc Natl Acad Sci USA 80:7581, 1983
6. Croce CM, Thierfelder W, Erikson J, Nishikura K, Finan J,
Lenoir GM, Nowell PC: Transcriptional activation of unrearranged
and untranslocated c-myc oncogene by translocatioh of a C X locus
in Burkitt lymphoma cells. Proc Natl Acad Sci USA 80:6922, 1983
7. Pelicci PG, Knowles DM, Magrath I, Dalla-Favera R: Chromosomal breakpoints and structural alterations of the c-myc locus differ
in endemic and sporadic forms of Burkitt lymphoma. Proc Natl Acad
Sci USA 83:2984, 1986
8. Joos S, Haluska FG. Falk MH, Henglein B, Hameister H,
Croce CM, Bornkamm GW: Mapping chromosomal breakpoints of
Burkitt’s t(8; 14) translocations far upstream of c-myc. Cancer Res
52:6547, 1992
9. Joos S, Martin HF, Lichter P, Haluska FG, Henglein B, Lenoir
GM, Bornkamm GW: Variable breakpoints in Burkitt lymphoma
cells with chromosomal t(8; 14) translocation separate c-myc and the
IgH locus up to several hundred Kb. Hum Mol Genet 1:625, 1992
10. Haluska FG, Russo G , Kant J, Andreef M, Croce CM: Molecular resemblance of an AIDS-associated lymphoma and endemic
Burkitt lymphomas: Implications for their. pathogenesis. Proc Natl
Acad Sci USA 86:8907, 1989
11. Ballerini P, Gaidano G , Gong JZ, Tassi V, Saglio G , Knowles
DM, Dalla-Favera R: Multiple genetic lesions in acquired immunodeficiency syndrome-related non-Hodgkin’s lymphoma. Blood
81:166, 1993
12. Zeidler R, Joos S, Delecluse HJ, Klobeck G, Vuillaume M,
Lenoir GM, Bornkamm GW, Lipp M: Breakpoints of Burkitt’s
lymphoma t(8;22) translocations map within a distance of 300 Kb
downstream of myc. Genes Chromosom Cancer 9:282, 1994
13. Shiramizu B, Magrath I: Localization of breakpoints by polymerase chain reactions in Burkitt’s lymphoma with 8; 14 translocations. Blood 75:1848, 1990
14. Le BeauMM: Detecting genetic changes inhuman tumor
cells: Have scientists “gone fishing?’” Blood 81:1979, 1993
15. Emmerich P, Loos P, Jauch A,Hopman AHW, Wiegant J,
Higgins M, White BN, van der Ploeg M, Cremer C, Cremer T:
Double insitu hybridization in combination with digitized image
analysis: A new approach to study interphas’echromosome topography. Exp Cell Res 181:126, 1989
16. Anastasi J, Vardiman JW, Rudinsky R, Patet M, Nachman J,
Rubin CM, Le Beau M: Direct correlation of cytogenetic findings
with cell morphology using insitu hybridization: An analysis of
suspicious cells in bone marrow specimens of two patients completing therapy for acute lymphoblastic leukemia. Blood 77:2456, 1991
17. Red T, Baldini A, Rand TC, Ward DC: Simultaneous visualization of seven different DNA probes using combinatorial fluorescence and digital imaging microscopy. Proc Natl Acad Sci USA
89:1388, 1992
18. Devilee P, Thieny RF, Kievits T, Kolluri R, Hopman AHN,
Willard HF, Pearson PL, Cornelisse CJ: Detection of chromosome
aneuploidy in interphase nuclei from human primary breast tumors
using chromosome-specific repetitive DNA probes. Cancer Res
485825, 1988
19. Cremer T, Lichter P,Borden J, Ward DC, Manuelidis L:
Detection of chromosomal aberrations in metaphases and interphase
tumor cells by in situ hybridization using chromosome-specific library probes. Hum Genet 80:235, 1988
20. Burke DT, Carle GF, Olson MV: Cloning of large segments
of exogenous DNA into yeast by means of artificial chromosome
vectors. Science 236:806, 1987
21. Larin Z, Monaco AP, Lehrach H: Yeast artificial chromosome
libraries containing large inserts from mouse and human DNA. Proc
Natl Acad Sci USA 88:4123, 1991
22. Sun LK, Showe LC, Croce CM: Analysis of the 3’ flanking
region of the human c-myc gene in lymphomas with the t(8;22) and
t(2; 8) chromosomal translocations. Nucleic Acids Res 14:4037, 1986
23. Haluska FG, Tsujimoto Y, Croce CM: The t(8; 14) chromosome translocation of the Burkitt lymphoma cell line DAUDI occurred during immunoglobulin gene rearrangement and involved the
heavy chain diversity region. Proc NatlAcadSciUSA84:6835,
1987
24. Henglein B, Synovzik H, Groitl P, Bornkamm GW, Hart1 P,
Lipp M: Three breakpoints of variant t(2;8) translocations in Burkitt’s lymphoma cells fall within a region 140 kilobases distal from
c-myc. Mol Cell Biol 9:2105, 1989
25. Haluska FG, Finver S, Tsujimoto Y , Croce CM: The t(8; 14)
chromssomal translocation occurring in B-cell malignancies results
from mistakes in V-D-J joining. Nature 324:158, 1986
26. Durst M, Croce CM, Gissmann L, Schwartz E, Huebner K:
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
VERONESE ET AL
2138
Papillomavirus sequences integrate near cellular oncogenes in some
cervical carcinomas. h o c Natl Acad Sci USA 84:1070, 1987
27. Czepulkowski BH, Bhatt B, Rooney DE: Basic techniques
for the prep,arationand analysis of chromosomes from bone marrow
and leukaemic blood, in Rooney DE, Czepulkowski BH (eds): Human Cytogenetics: Constitutional Adalysis in Lymphocyte Culture,
vpl 1. New York, NY, Oxford, 1992, p 1
28. Wiman KG, Clarkson B, Hayday AC, Saito H, Tonegawa S,
Hayward WS: Activation of a translocated c-myc gene: Role of
structurd alterations in the upstream region. ProcAcad Sci USA
81:6798, 1984
29. Erikson J, ar-Rushdi A, Drwinga H, Nowell PC, Croce CM:
Transcriptional activation of the translocated c-myc oncogene in
Burkitt lymphoma. Proc Acad Sci USA 80:820, 1983
30. Cross NC, Melo JV, Feng L, Goldman JM:An optimized
multiplex polymerase chain reaction (PCR) for detection of BCRABL fusion mRNAs in haematological disorders. Leukemia 8:186,
1994
_
__.
31, Gribben JG, Neuberg ,D, FreedmanAS, Gimmi CD, Pesek
KW, Barbet M, Saporito L, Woo SD, Coral F, Spector N, Rabinowe
SN, Grossbard ML, Ritz J, Nadler LM: Detection by polymerase
chain reaction of residual cells with the bcl-2 translocation is associat& with increased risk of relapse after autologous bone marrow
transplantation for B-cell lymphoma. Blood 81:3449, 1993
32. Anastasi J, Thangavelu M, Vardiman J W , Hooberman AL,
Bian ML, Larson RA, Le Beau MM: Interphase cytogenetic analysis
detects minimal residual disease in a case of acute lymphoblastic
leukemia and resolves the question of origin of relapse after allogeneic bone marrow translantation. Blood 77:1087, 1991
33. Bentz M, Cabot G, Moos M, Speicher MR, Ganser'A, Lichter
P, Dohner H: Detection of chimeric BCR-ABL genes on bone marrow
samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 83:1922, 1994
34. Rowley JD, Diaz MO, Espinosa R 111, Patel YD, van Melle
E, Ziemin S, Taillon-Miller P, Lichter P, Evans GA, Kersey JH,
Ward DC, Domer PH, LeBeauM:
Mapping chromosome band
1 lq23 in human acute leukemia with biotinylated probes: Identification of 1lq23 translocation breakpoints with a yeast artificial chromosome. Roc Natl Acad Sci USA 87:9358, 1990
35. Taniwaki M, Matsuda F, Jauch A, Nishida K, Takashima T,
Tagawa S, Sugiyama H, Misawa S, Abe T, Kashima K: Detection
of 14q32 translocations in B-cell malignancies by in situ hybridization with yeast artificial chromosome clones containing the human
IgH gene locus.
1994
T, Lengauer c, Cremer T, Wiegmt J, Rasp AK, van
36.
der
M, Groitl p, LiPP M: Specific metaphase and interphase
detection of the breakpoint region in8q24 of Burkitt lymphoma cells
by triple-color fluorescence in situ hybridization. Genes Chromosom
Cancer 4:69, 1992
83:29623
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1995 85: 2132-2138
Detection of myc translocations in lymphoma cells by fluorescence in
situ hybridization with yeast artificial chromosomes
ML Veronese, M Ohta, J Finan, PC Nowell and CM Croce
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