1. No category

From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
1993 81: 122-127
Analysis of the expression of the hybrid gene bcl-2/IgH in follicular
lymphomas
P Soubeyran, F Cabanillas and MS Lee
Updated information and services can be found at:
http://www.bloodjournal.org/content/81/1/122.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
Analysis of the Expression of the Hybrid Gene bcl-2/IgH
in Follicular Lymphomas
By Pierre Soubeyran, Fernando Cabanillas, and Ming Sheng Lee
To investigate the clinical and biologic significance of the
circulatingt(l4; 18)-carryingcells in follicular lymphoma (FL)
patients, we analyzed the mbr/JH junction of the hybrid
bcl-2/lgH gene simultaneously at the DNA and RNA levels
by polymerase chain reaction (PCR) in 37 peripheral blood
samples from 37 patients in different remission status: 4
before treatment, 8 during treatment, and 25 in complete
remission (CR). Of these 37 patients, 22 were positive either at the DNA or RNA level (8 with active disease and
1 4 in CR). Among these positive patients, RNA was more
often negative for patients in CR (9 of 1 4 [64%]) than for
patients with active disease (2 of 8 [25%]; Fisher's exact
test, P = .09). Among the 1 4 patients in CR with residual
disease, 2 of 5 with RNA positivity relapsed, whereas 1 of
9 with RNA negativity and DNA positivity relapsed with a
median follow-up after sample collection of 8 months
(range, 4 to 1 8 months). Simultaneous analysis of the bcl2/lgH gene at the DNA and RNA level showed heterogeneous patterns of PCR positivity in regardsto the evaluation
of the biologicactivity of the t(l4; 18)-canyingcells. A larger
study and long-term follow-up will help in determining
whether the expression patterns in turn reflect the functional status of disease activity in FL patients.
0 1993 by The American Society of Hematology.
F"
the marker could either be benign or quiescent, so that there
would be no imminent threat of disease recurrence. However,
if they do represent a residual malignant clone with high
proliferative activity, the disease would be expected to relapse
early. Such speculations have led us to pursue whether there
are any means to examine the biologic activities of the minimal residual t( 14; 18)-carrying cells, such as the expression
of the chimeric bcl-2/IgH messenger RNA (mRNA). In this
report, we demonstrated the feasibility of applying PCR to
the detection of extremely small amounts of chimeric bcl-2/
IgH mRNA produced from circulating t( 14; 18)-carrying cells.
Consequently, we also studied the clinical values of this
RNA PCR assay in FL.Peripheral blood samples were collected and analyzed before treatment, during therapy, and
in remission. Peripheral blood appears to be a good choice
for residual disease detection because it is easily accessible
and it has been previously shown that at least 80% of B-cell
lymphomas have monoclonal circulating cells at initial presentation. l 4
LLICULAR lymphomas (FL) represent a large part of
the non-Hodgkin's lymphomas (NHL).' They are characterized by the t( 14; 18) chromosomal translocation that
juxtaposes the bcl-2 gene with the Ig heavy chain gene (IgHX2
which, in turn, results in the overexpression of a chimeric
bcl-2/IgH me~sage.'.~
Because the chromosomal breakpoint
falls outside the translated portion of the bcl-2 gene, the protein product is identical with the normal bcl-2 p r ~ t e i n . ~
Recently, Hockenbery et a16 showed that the bcl-2 protein
is able to block programmed cell death (apoptosis). Consequently, the rearrangement of bcl-2 with IgH leads to longer
survival of the concerned cells that allows secondary transforming events to occur.
Although clinical complete remission (CR) is easily
achieved in low-grade FL, the relapse rate is high, raising the
problem of minimal residual disease resistant to systemic
treatment.' Detection of minimal residual disease in F'L patients in CR has been very actively pursued over the last
years. Different methods have been used: detection of Ig gene
rearrangement by Southem blotting: clonal excess methods:
and polymerase chain reaction (PCR)."." Whereas Southem
blotting and clonal excess methods allow the detection of 1
to 10 abnormal cells among 100 normal cells, PCR is able
to recognize one abnormal cell among 100,000 normal
cells.'o," However, recent data tend to show that this minimal
residual disease is not clearly related to an increased risk of
relapse in FL.I2,I3All these methods provide information on
the presence or absence of putative tumor markers characteristic of the disease, but they fail to examine the functional
status of the residual disease. The circulating cells carrying
From the Department of Laboratory Medicine and the Department
of Hematology, University of Texas MD Anderson Cancer Center,
Houston.
Submitted March 23, 1992; accepted August 25, 1992.
Supported by National Institutes of Health Grant CA49443. M.S.L.
is a special fellow of Leukemia Society of America.
Address reprint request to Pierre Soubeyran, MD, Fondation Bergonit?, 180, rue de Saint-Gen;s, 33076 Bordeaux Cedex, France.
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 1993 by The American Society of Hematology.
0006-4971/93/8101-0010$3.00/0
122
MATERIALS AND METHODS
DNA and RNA extraction. High molecular weight DNA and total
cellular RNA were simultaneouslyextracted from blood samples using
a method previously described15 using guanidine thiocyanate, cesium
chloride, and ultracentrifugation.
DNase treatment of RNA. The total RNA samples prepared as
described above were further treated by DNase I (RNase free) in a
total reaction volume of 35 pL (50 mmol/L Tris, pH 7.8; 1 mmol/L
EDTA, pH 8.0 I O mmol/L MgCI2, 1 mmol/L dithiothreitol, 40 U
RNasin [Promega, Madison, WI]; 60 U DNase 1 RNase-free [Boehringer Mannheim Biochemica, Indianapolis, IN]). The reaction was
incubated at 37°C for 1 hour and then heated at 95°C for 5 minutes
and immediately cooled on ice.
Reverse transcription of RNA. One to 3 pg of these DNase-treated
RNA was submitted to reverse transcription. The reaction was performed in a total reaction volume of 40 pL in the following conditions:
50 mmol/L Tris-HCI, pH 8.3; 75 mmol/L KCI; 3 mmol/L MgCI2;
1 mmol of each d N T P 400 nmol/L random primers (Boehringer
Mannheim Biochemica); 5 mmol/L dithiothreitol; 40 U RNasin
(Promega); and 400 U MMLV reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD) at 37°C overnight.
PCR methods. The sequences of the different oligonucleotide
primers used are outlined in Table 1. The primer mbr3+ is derived
from the sequence 5' to the major breakpoint clustering region (mbr)
of the bcl-2 gene.16 JH- primer is derived from the consensus sequence that has been described at the 3' end of the six different JH
Blood, Vol81, No 1 (January l), 1993:pp 122-127
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
123
EXPRESSION OF b~l-2/lgHIN FOLLICULAR NHL
Table 1 . Oligonucleotide Primers and Probes’ Sequences
Mbr3+
JH18q21 probe
Raf 8 i
Raf 9Raf 9 Drobe
5 TTT GAC CTT TAG AGA GTT GCT TTA CG 3
5 ACC TGA GGA GAC GGT GAC C 3
5 CAC AGA CCC ACC CAG AGC CC 3
5 GAT GCA ATT CGA AGT CAC AGC G 3‘
5 TTT TCT CCT GGG TCC CAG ATA 3
5’ GTC CAG TAG CCC CAA CAA TCT G 3
segments of the IgH gene.” These two primers were able to amplify
the crossover site sequence of the t( 14; 18) within the mbr region in
approximately 60% to 70% of FL patients. The length of the fragments
amplified ranged from approximately 150 to 400 bp. The same primers were used for DNA and cDNA amplification. Because this set of
primers does not span any intron, the length of the PCR product is
the same in the same patient for DNA and cDNA. Therefore, it is
extremely important to rule out the problem of DNA contamination
in the RNA sample. Removal of the DNA contamination was performed by DNase I treatment as described above. The effectiveness
of removing DNA contamination was confirmed by PCR of c-raf-1
gene. Primer derived from exon VI11 of c-raf-1 gene (raw+) and
primer derived from exon IX of c-raf-I gene (raf9-) span an intron
110 bp in length.’’ The PCR product will be 148 bp in length on
cDNA amplifiatsand 258 bp in length in case of DNA contamination.
cDNA amplification of the c-raf-I gene also allows us to directly
evaluate the effectiveness of reverse transcription. Because the
expression of c-raf I is ubiquitous,” the PCR product represents the
whole pool of RNA.
For each patient sample, three different PCR reactions were simultaneously performed in three different tubes with the mbr3+ and
JH- primers: 1 pg of DNA, cDNA generated from 0.6 to 1 pg of
RNA using random hexamers, I pg of RNA without reverse transcription (as negative control to rule out DNA contamination).
PCR amplification was performed as a modification of the method
previously described’’ for 45 cycles using a thermal cycler (Perkin
Elmer Cetus, Nonvalk, CT). Patients were considered positive when
they were positive either at the DNA or RNA level.
We followed the recommendations of Kwok and Higushi2Oconcerning control of contamination. Furthermore, because the breakpoint of each patient is different, the length of the PCR product is
different. In this way, PCR contamination is easier to identify than
within other models. Finally, we always ran two negative controls
(negative DNA and no template) to confirm that our PCR reaction
was free of false-positivitysecondary to contamination.
Southern blot analysis of the PCR products. Fifteen percent of
PCR products were size fractionated in a 2% Nu Sieve gel (FMC
Bioproducts, Rockland, ME) and then transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH), as described by Southern.” Membranes were then hybridized with a 5’ end radiolabeled
oligonucleotide probe 18q21 (Table 1) at 42°C overnight. Washing
was performed in 2 SSPE/O.170SDS at room temperature for 1 hour.
Autoradiography was performed against a single intensifying screen
at -70°C for 72 hours.
Patients. Thirty-seven blood samples obtained from 37 patients
were analyzed, including 12 with active or persistent disease (4 before
and 8 during treatment) and 25 during CR. The main characteristics
of these two groups of patients are outlined in Table 2. All patients
except one were treated with chemotherapy using either CHOP Bleo22
or other regimens with or without radiotherapy. The exceptional
patient received exclusive radiation only.
The duration of CR has been computed from the date of the first
negative work-up (including all initially positive exams) to the date
of the sample collection.
RESULTS
Removal of the DNA contamination from the RNA samTo ensure that our RNA samples were free of DNA
contamination, we used DNase I treatment that was able to
completely remove DNA contamination of intentionally
contaminated RNA. Two RNA samples were analyzed, one
contaminated by I pg of DNA per 1 pg of RNA and the
other by I O ng of DNA per I pg of RNA. After DNase I
treatment, reverse transcription PCR (RT-PCR) of c-raf- 1
gene was performed. As shown in Fig 1, it is efficient in removing the DNA contamination of the RNA while maintaining RNA quality. This allowed us to further study patients
samples.
Vererification of the intactness of RNA samples and efectiveness of reverse transcription. All RNA samples were reverse transcribed using random hexamers as primers and then
tested by the c-raf-1 amplification. In all cases, amplification
of the c-raf- 1 cDNA showed an adequate amount of product,
confirming that the RNA quality was good enough for PCR
amplification and that the reverse transcription worked correctly.
Confirmation of adequate PCR eficiencies in patients’
samples analyses. Sensitivity of our PCR assay was tested
on serially diluted DNA samples; from 1 pg to 1 pg of positive
DNA was diluted in I pg of negative DNA. We could easily
detect the hybrid bcl-2/IgH junction in samples containing
I O pg or more of positive DNA (data not shown). When
patients samples were amplified, two positive control samples
(10 ng and 100 pg) were also amplified in parallel to ensure
that PCR was performed at its optimal efficiency.
P C R amplijication of the chimeric bcl-Z/IgH DNA and
RNA in F L patients’ samples. Of the 31 patients (4 pretreatment, 8 during treatment, and 25 in CR), 16 were positive
at the DNA level (43%)and I 1 at the RNA level (30%).Considering DNA and RNA results together, 22 patients were
positive (60%): I I with DNA positivity/RNA negativity, 5
with DNA positivity/RNA positivity, and 6 with DNA negativity/RNA positivity (Table 3 and Fig 2).
ples.
Table 2. Main Characteristics of the Patients
Age (VI
Mean
Range
Sex
Male
Female
Stage
I
II
111
IV
Pathology
FSC
FM
FLC
Presence of Disease
(12 patients)
(25 patients)
48
26-72
53.5
28.5-77
CR
6
6
16
9
1
1
3
7
3
5
5
12
11
1
0
16
4
5
Abbreviations: FSC, follicular small cleaved; FM, follicular mixed; FLC,
follicular large cell.
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
124
SOUBEYRAN, CABANILLAS, AND LEE
Resiilts of samples.fiom I2 patients collected either before
Eight of these I2 patients were positive
(66%)either by D N A or RNA testing (Table 3). D N A positivity was detected in 6. whereas RNA FTR allowed two more
positive diagnoses.
Of these 12 patients, 4 were pretreatment samples. All 4
were positive either by D N A or RNA. One patient showed
a strong signal for D N A without any expression of the hybrid
message of bcl-2 at the RNA level.
Eight samples were collected during treatment (after 2 to
7 months of treatment by chemotherapy or a-interferon [aIFN]). Four patients were positive either at the D N A or RNA
level. Two of these presented a strong expression of the hybrid
message of bcl-2 with either a weaker band (patient B, Fig 2)
or not any detectable band at the D N A level (patient E. Fig
2). The remaining 2 patients presented a strong signal at the
D N A level with either a weak expression in one case (patient
C, Fig 2) or no expression at all at the RNA level. These 4
patients were treated with combination chemotherapy and
a-IFN.
Resirhs qf 25 samp1e.s collected hiring CR. Fourteen of
these were positive either at the D N A or RNA level, with IO
detectable at the D N A level and 5 at the RNA level (Table
3). Ofthe IO DNA-positive patients in CR, only one presented
a detectable level of expression of hybrid bcl-Z/lgH message
by PCR. Four patients in CR showed only RNA positivity.
The results were further correlated with the duration of remission (Table 4). Eight of 13 patients (61.5%) in CR less
than 3 years showed PCR positivity, including 5 with RNA
negativity. Six of IO patients (60%)in CR for 3 to 5 years
had PCR positivity, including 4 with RNA negativity.
Table 3. Detection of the Chimeric bcl-2/lgH Junction
at the DNA and RNA Level
or during treatment.
M
271/281 bp
234 bp
194 bp-
-
-
118 bp-
1
2
3
4
Positive
DNA+/RNADNA+/RNA+
DNA-/RNA+
Negative
DNA-/RNA-
Before Therapy
During Therapy
CR
4 (100)
1
2
1
4 (50)
1
2
1
14 (56)
9
1
4
0
4 (50)
11 (44)
Percentages are in parentheses.
Our results were also correlated with the pretreatment
clinical Ann Arbor stage. Of the eight patients in stages I and
11, four were positive, including two at the D N A level only,
one at both D N A and RNA level, and one exclusively at the
RNA level. None of these localized stages relapsed.
Among 17 patients in stages 111 and IV, IO were positive.
including 7 exclusively at the D N A level and 3 exclusively
at the RNA level.
Three of these 25 patients relapsed 4 to IO months after
the sample collection. All three were of follicular small cleaved
cell histology. All of them presented residual disease at the
time of PCR analysis. including 2 with hybrid bcl-2/IgH RNA
expression (Table 5). All the other patients are still in CR,
with a median follow-up after sample collection of 12.5
months (range, 0 to 32.5 months).
Median follow-up computed from date of diagnosis to date
last known alive was49.5 months(range, 12.5 to 91 months)
for the 25 CR patients and 15 months (range, 4 to 42.5
months) for the 12 with active disease.
5
6
Fig 1. Set-up of the DNase I
treatment of RNA. Three percent NuSieve gel stained with
ethidium bromide. Lane M corresponds to marker 174-Hee
111 digested. Lanes 1 and 3 represent, respectively, RNA samples contaminatedwith 1 pg and
1 0 ng of DNA per 1 pg of RNA.
Lanes 2 and 4 represent the
same samples after treatment
by DNase I for 1 hour at 37°C.
In all cases, 1 pg of RNA was
used as template for reverse
transcription. Lane 5 is the positive DNA control (1 pg of DNA)
and lane 6 the negative control
(no template). The DNA band
(258 bp) is visible in lanes 1 and
3, whereas it is not visible in
lanes 2 and 4, demonstrating
the efficiency of the treatment.
RNA bands are visible in lanes
1 to 4, showing the integrity of
RNA as well as the efficiency of
reverse transcription.
*
- 258 bp
-
148bp
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
125
EXPRESSION OF b~l-2/lgHIN FOLLICULAR NHL
B
A
1 2 3
-
bP
1
2
D
C
3
1
2
3
1
2
E
3
1
2
3
310 bp 194 bp Fig 2. Results of the PCR for five dRerent patients representative of dRerent situations. Hybridization with 18q21 probe. Lanes 1
represent DNA PCR: lanes 2 represent cDNA PCR; and lanes 3 represent PCR of RNA without reverse transcription. Patients A, 6,C, and
E still had presence of disease at the time of sample collection, whereas patient D was in CR at this time. Patients A and B represent the
common case of strong RNA expression with detectable DNA signal, whereas patient E represents detection at the RNA level with no
detection on DNA. Patient C shows a good DNA signal and a faint RNA band, whereas patient D shows DNA but no RNA band.
DISCUSSION
Analysis of the pattern of expression of the hybrid message
of bcl-Z/lgH within circulating residual cells of patients with
t( 14; 18) to our knowledge has not been reported. PCR is well
suited to this kind of study because it provides high sensitivity
and can be performed in low amounts of even partially degraded RNA. Because the sequence at the juncture of mbr/
IgH region is the same at the DNA and the mRNA levels.
the most pertinent technical problem in the mRNA PCR
assay for the t( 14: 18) is DNA contamination. We have undertaken multiple precautions to control this problem of
DNA contamination in RNA, which is commonly observed
in the extraction of RNA. First, RNA was cleaned from DNA
through a DNase I treatment. Secondly, the efficiency of DNA
removal was assessed in two ways: c-rufil amplification and
negative RNA control (without any reverse transcription).
The c-rup I amplification allowed us to assess both the quality
of the RNA and the efficiency of our reverse transcription
reaction. This information ensured the reliability of our mbr3JH RNA PCR because the same pool of cDNA was used for
both PCRs.
We also took special precautions to control the risk of
contamination from previously amplified positive samples.
Indeed, the search for residual disease is highly exposed to
this kind of risk.23The bcl-Z/lgH model is easier than the
others in this aspect because it provides an additional control
by the size of the PCR product, which is variable from patient
to patient. We used multiple negative controls in each reaction
and used highly diluted positive controls to limit the production of positive PCR product. Finally, we did not try to
Table 4. Positive Results and Duration of CR
PCR-Positive
Durationof
Remission
DNA+/
RNA-
DNA+/
RNA+
DNA-/
RNA+
PCRNegative
Total
Less than 3 yr
3 to 5 yr
More than 5 yr
5
4
0
0
1
3
1
0
5
4
2
13
10
2
0
increase the PCR efficiency by either increasing the amount
ofTaq polymerase or using boosted PCR." We preferred the
use of hybridization with an internal probe to increase the
specificity of our PCR assay. Around 60% to 70% of FL
patients with the t( 14: 18) have the breakpoints falling
within the mbr region.24Our PCR assay is confined to
examining this subpopulation of patients. We observed
66% of positive results among the 12 treatment samples
and 56% among the 25 CR samples. Among 27 patients
with follicular small cleaved cell histology, 17 were positive
(63%):among five follicular mixed cell samples, three were
positive (60%);and among five follicular large cell samples,
two were positive.
Simultaneously analyzing the mbr/JH junction at the
DNA and R N A level, we have observed four patterns of
results: ( I ) negative at both DNA and R N A level; (2) positive DNA and negative RNA; (3) negative DNA and positive RNA: (4) positive DNA and RNA. No firm conclusion
can be drawn from the double-negative samples because
we do not know whether this represents a tumor without
Table 5. Patients in CR Results of the mbr3-JH PCR Assay
CR
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
DNA
RNA
+
-
+
+
+
-
+
+
-
+
-
+
-
+
+
-
-
+
+
+
+
+
Delay Sample/
Relapse
Duration
(mol
Relapse
29
3
4
4
50
56
3
40
56
50
40
8
31
33
NO
No
No
Yes
NO
NO
No
NO
NO
No
Yes
Yes
No
NO
Abbreviation: F/U, follow-up.
0"
10
4
4.5
F/U Duration
From Sample
(mol
32.5
16
23
23
14.5
10
16
7.5
5.5
13
13
4.5
9
11.5
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
SOUBEYRAN, CABANILLAS, AND LEE
126
3. Graninger WB, Set0 M, Boutain B, Goldman P, Korsmeyer
a breakpoint at the mbr region of bcl-2 or the absence of
SJ: Expression of bcl-2 and bcl-2-Ig fusion transcripts in normal and
residual disease in patients with bcl-2 rearrangement. Furneoplastic cells. J Clin Invest 80: 15 12, 1987
thermore, PCR negativity by no means indicates complete
4. Set0 M, Jaeger U, Hockett RD, Graninger W, Bennett S, Goldabsence of t( 14; I8)-bearing cells, which could be too few
man P, Korsmeyer SJ: Alternative promoters and exons, somatic
to be detected by PCR. Our current knowledge leads us to
mutation and deregulation of the bcl-2-Ig fusion gene in lymphoma.
expect a strong expression of the hybrid message bcl-2/
EMBO J 7:123, 1988
IgH.’ At the PCR level, we indeed frequently observe a
5. Tsujimoto Y , Croce C Analysis of the structure, transcripts,
stronger signal for RNA than for DNA. That is what we
and protein products of bcl-2, the gene involved in human follicular
observed 3 out of 4 times in pretreatment samples (75%))
lymphoma. Proc Natl Acad Sci USA 83:5214, 1986
2 out of 4 times in samples collected during treatment
6. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ: Bcl-2 is an inner mitochondrial membrane protein that
(50%),and 5 out of 14 times in the positive samples among
blocks programmed cell death. Nature 348:334, 1990
cases in CR (36%).
7. Lister T A The management of follicular lymphoma. Ann Oncol
One of our pretreatment samples exhibited bcl-2 rear2:131, 1991 (suppl2)
rangement at the DNA level without any RNA expression
8. Homing SJ, Galili N, Cleary M, Sklar J: Detection of nonof the same kind. This raises the question as to whether the
Hodgkin’s lymphomas in the peripheral blood by analysis of antigen
hybrid bcl-2/IgH message could be downregulated by either
receptor gene rearrangements: Results of a prospective study. Blood
extrinsic or probably intrinsic factors to the tumor. One case
75:1139, 1990
of absence of expression of the chimeric message of bcl-2/
9. Johnson A, Cavallin-Stahl E, Akerman M: The significance of
IgH has been described.25However, it represents a unique
B-clonal excess in peripheral blood in patients with non-Hodgkin’s
t(8; 14; 18) that juxtaposed the c-myc gene to bcl-2 and IgH.
lymphoma in remission. Ann Oncol 2:99, 1991 (suppl 2)
Finally, previous analyses3s4clearly show that the absence of
10. Lee MS, Chang KS, Cabanillas F, Freireich EJ, Trujillo JM,
expression of bcl-2/IgH in cases of t( 14; 18) is expected to
Stass SA: Detection of minimal residual cells carrying the t( 14; 18)
by DNA sequence amplification. Science 237: 175, 1987
occur infrequently.
11. Crescenzi M, Set0 M, Herzig GP, Weiss PD, Griffith RC,
Among the four positive samples drawn during treatment,
Korsmeyer SJ: Thermostable DNA polymerase chain amplification
two exhibited an absence or a weak expression of the bcl-2/
of t(14; 18) chromosome breakpoints and detection of minimal reIgH message. One patient was being treated with CHOPsidual disease. Proc Natl Acad Sci USA 85:4869, 1988
Bleo combination and the other was being treated with a12. Price CGA, Meerabux J, Murtagh S, Cotter FE, Rohatiner
IFN.It raises the possibility that at least one of the therapeutic
AZS, Young BD, Lister TA: The significanceof circulating cells caragents may downregulate the RNA expression. This is posrying t( 14; 18) in long remission from follicular lymphoma. J Clin
sible with most of the drugs used in the CHOP Bleo comOncol 9: 1527, 1991
bination and also with a-IF” because it is already known to
13. Lee M, Cabanillas F, Freireich E, Trujillo J, Stass S: Minimal
turn off the c-myc message in some tumors.26This possibility
residual circulating cells carrying the t( 14; 18) are present in patients
is also supported by the results of the samples obtained during
with follicular lymphoma or diffuse large cell lymphoma in long term
remission. Blood 72:247a, 1988 (abstr, suppl 1)
CR that showed RNA expression only in 5 of 14 positive
14. Smith BR, Weinberg DS, Robert NS, Towle M, Luther E,
samples.
Pinkus G, Ault KA: Circulating monoclonal B lymphocytes in nonDetection of residual bcl-2 rearranged cells in FL has not
Hodgkin’s lymphoma. N Engl J Med 31 1:1476, 1984
shown any predictive value on the risk of relapse. Even long15. Sambrook J, Fritsch EF, Maniatis T Extraction of RNA with
term remission patients with a low risk of relapse are freguanidinium thiocyanate followed by centrifugation in cesium chloquently p~sitive.’~,’’
Two hypotheses can be drawn”: either
ride solutions, in Ford N, Nolan C, Ferguson M (eds): Molecular
the detected cells are t( 14; 18)-bearing cells that are not fully
Cloning. A Laboratory Manual (ed 2). Cold Spring Harbor, Cold
malignant [bearing only the t( 14; 18), but not the secondary
Spring Harbor Laboratory, 1989, p 7.19
genetic events leading to malignant t r a n s f o r m a t i ~ n ~or
~ . ~ ~ ] 16. Cleary ML, Smith SD, Sklar J: Cloning and structural analysis
they represent tumor cells that are quiescent (temporarily or
of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript
not). RNA PCR could be informative in this regard because
resulting from the t(14; 18) translocation. Cell 47:19, 1986
it examines the expression of the chimeric gene that plays an
17. Ravetch JV, Siebenlist U, Korsmeyer S, Waldmann T, Leder
P Structure of the human immunoglobulin p locus: Characterizaimportant role in the pathogenesis of the cells carrying the
tion of embryonic and rearranged J and D genes. Cell 27:583,
t( 14; 18). Yet, a large number of patients and longer follow1981
up are obviously necessary to assess the predictive value of
18. Bonner TI, Kerby SB, Sutrave P, Gunnel1 MA, Mark G, Rapp
this test in regards to risk of relapse.
REFERENCES
1. Hoerni B, Trojani M, Eghbali H, Bonichon F, Coindre JM,
Garraud 0, Hcemi-Simon G: Distinct entities among low grade nonHodgkm’s lymphomas. Analysis of a series of 377 cases. Eur J Cancer
23:1889, 1987
2. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM:
Cloning of the chromosome breakpoint of neoplastic B cell with the
t( 14; 18) chromosome translocation. Science 226: 1097, 1984
UR: Structure and biological activity of human homologs of the raf/
mil oncogene. Mol Cell Biol 5:1400, 1985
19. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn
GT, Mullis KB, Ehrlich HA: Primer directed enzymatic amplification
of DNA with a thermostable DNA polymerase. Science 239:487,
1988
20. Kwok S, Higushi R: Avoiding false positive with PCR. Nature
339:237, 1989
21. Southern EM: Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol 98:503, 1975
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
EXPRESSION OF bcl-l/lgH IN FOLLICULAR NHL
22. Rodriguez V, Cabanillas F, Burgess MA, McKelvey EM, Valdivieso M, Bodey GP, Freireich EJ: Combination chemotherapy
(“CHOP-Bleo”) in advanced (non-Hodgkin) malignant lymphoma.
Blood 49:325, 1977
23. Negrin RS, Blume KG: The use of the polymerase chain reaction for the detection of minimal residual malignant disease. Blood
78:255, 1991
24. Weiss LM, Warnke RA, Sklar J, Cleary ML: Molecular analysis
of the t(14; 18) chromosomal translocation in malignant lymphomas.
N Engl J Med 317:1185, 1987
25. Kiem HP, Nourse J, Saltman DL, Blume KG, Cleary M L
Concurrent activation of c-myc and inactivation of bcl-2 by chromosomal translocation in a lymphoblastic lymphoma cell line. Oncogene 5 : 18 15, 1990
127
26. Einat M, Resnitzky D, Kimchi A: Close link between reduction
of c-myc expression by interferon and GO/GI arrest. Nature 313:
597, 1985
27. Hugues TP, Goldman JM: Biological importance of residual
leukemic cells after BMT for CML: Does the polymerase chain reaction help? Bone Marrow Transplant 5:3, 1990
28. McDonnell TJ, Nunez G, Platt FM, Hockenbery D, London
L, McKearn JP, Korsmeyer SJ: Deregulated bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin
M and D-expressing B-cell population. Mol Cell Biol 10:1901,
1990
29. Fialkow PJ, Janssen JWG, Bartram CR: Clonal remissions in
acute nonlymphocytic leukemia: Evidence for a multistep pathogenesis ofthe malignancy. Blood 77:1415, 1991