1. No category

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1995 86: 2312-2320
Clonal relationship between lymphocytic predominance Hodgkin's
disease and concurrent or subsequent large-cell lymphoma of B
lineage
RS Wickert, DD Weisenburger, A Tierens, TC Greiner and WC Chan
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Clonal Relationship Between Lymphocytic Predominance Hodgkin’s Disease
and Concurrent or Subsequent Large-Cell Lymphoma of B Lineage
By R.S. Wickert, D.D. Weisenburger, A. Tierens, T.C. Greiner, and W.C. Chan
The occurrence of a large-cell lymphoma (LCL) concurrent
with or subsequent t o lymphocytic predominanceHodgkin’s
disease (LPHD) is well documented. Given the well-characterized B-cell nature of the Reed-Sternberg cell variants in
LPHD, there may bea clonal relationship between theLPHD
and the associated B-cell LCL. In this study, we adapted a
highly sensitive, clonospecific assay t o test whether the
clone comprising the LCL exists in the corresponding LPHD
tumor. Nine cases meeting the histologic
criteria of nodular
LPHD and B-cell LCL were identified, reviewed, and studied.
Initially, c l o n a l i i of bothlesions was assessedusing consensus primers t o conserved regions in the IgHvariable (framework 111) and joining region genes in a polymerase chain
reaction (PCR) assay. The PCR assay detected a clonal B-cell
population in five of the LCLs, whereas analysis of eight
cases of LPHD did not detect a dominant clone. Clonal products from theLCL were then sequenced, and clonospecific
oligonucleotides were designed from theunique nucleotide
sequence encoding the complementarity-determiningregion-Ill. These were then used as primers and/or probes in
sensitive PCR-based assayson thecorresponding LPHD tumors. In two cases, the clonospecific assay showed that the
LPHD and LCL shared a common clone that was further confirmed bysequence analysis. This finding provides genotypic
evidence that, at least in some cases, the LCL represents a
clonal progression of LPHD.
0 1995 b y The American Society of Hematology.
I
flicting results,””’ and Southern blot analysis has seldom
shown a clonal immunoglobulin gene rearrangement.I6One
recent study reported clonal immunoglobulin heavy chain
(IgH) gene rearrangements in a substantial percentage of
cases of LPHD by the polymerase chain reaction (PCR)
analysis.” However, we have not been able to confirm monoclonality in the L&H cells by single-cell analysis.’*
Over the years, small series of cases in which a large-cell
lymphoma (LCL) occurred concurrent with or subsequent to
a diagnosis of LPHD have been
The majority of such LCLs have been shown tobelongto
the Blineage using immunohistochemical and/or molecular techn i q ~ e s . ~ ”It- ’ has
~ been suggested thatLPHD represents a
polyclonal lymphoproliferation that originates in the germinal centers and that the development of B-LCL represents a
histologic pr~gression.’’~~~
We report our investigation of the relationship between
LPHD and subsequent or concurrent B-LCL. The hypothesis
that a small B-cell clone, most likely derived from an L&H
cell, undergoes transformation and progresses to B-LCL was
tested using sensitive clonospecific (CS) molecular techniques.
N 1944, JACKSON AND PARKER’ described a subset
of Hodgkin’s disease (HD), called paragranuloma, in
which the lymph node structure was obliterated by an infiltrate with an abundance of lymphocytes. In 1966, Lukes and
Butlef renamed paragranuloma as HD of the lymphocytic
and histiocytic (L&H) type and further divided it into two
subtypes, nodular and diffuse, taking into consideration the
growth pattern of the tumor. These two subtypes were later
combined at the Rye conference3 into the lymphocytic predominance HD (LPHD), which comprises approximately S%
of newly diagnosed cases of HD.4
Classical Reed-Sternberg (RS) cells are rare in LPHD,
whereas the RS variant known as the L&H cell is relatively
frequent and usually present singly or in small clusters. A
nodular growth pattern is easily appreciated in about 80%
of the cases, and the nodules bear a resemblance to the large,
altered germinal centers called progressively transformed
germinal centers (PTGC), which have been reported in
lymph nodes biopsied before, after, or concurrent with the
diagnosis of LPHD.’”
The L&H cells consistently express a number of B cellassociated molecules, such as CD20, CD22, mb-l, and cytoplasmic J-chain.‘”’ However, attempts to demonstrate clonality immunohistochemically by light chain restriction have
generally been unsuccessful, although Schmid et all2 have
reported light chain restriction in S of 19 cases of LPHD.
Recent studies using in situ hybridization to detect cytoplasmic kappa and lambda mRNA have also yielded conFrom the Department of Pathology and Microbiology, University
of Nebraska Medical Center, Omaha, NE.
Submitted March 6, 1995; accepted May 3, 1995.
Supported in part by National Institutes of Health Grant No.
CA36727.
Address correspondence to Wing C. Chan, MD, Department of
Pathology and Microbiology, University of Nebraska Medical Center, 600 S 42nd St, Omaha, NE 68198-3135.
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 1995 by The American Society of Hematology.
0006-4971/95/86~-0005$3.00/0
2312
MATERIALS AND METHODS
Patient Population and Pathology Review
Nine patients with a diagnosis of LCL subsequent to or concurrent
with LPHD were identified in the files of the Nebraska Lymphoma
Registry and the consultation files of two of us (W.C.C., D.D.W.).
All biopsies and pathology records were reviewed to confirm the
diagnoses, and the paraffin blocks were obtained from the referring
institutions. In one patient, no paraffin blockrepresentative of LPHD
was available for study.
DNA Preparation
A modification of the method described by Wright and Manos2’
was used to extract DNA from the paraffin-embedded, formalin- or
BS-fixed tissue. Five-micrometer sections of tissue were dewaxed
with xylene and washed in absolute ethanol. The sample was then
digested overnight at 50°C in 1X PCR buffer (50 mmom KCl, 10
mmol/L Tris-HC1 pH 8.3, 0.1 mg/mL gelatin, 0.45% Nonidet P40,
and 0.45% Tween 20) containing 200 pg/mL of proteinase K. Finally, the proteinase K was inactivated by a IO-minute incubation
at94°C. For tissues fixedin B5, the procedure was modified to
Blood, Vol 86, No 6 (September 15), 1995: PP 2312-2320
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LYMPHOMA
2313
Table 1. Sequence of Oligonucleotide Primers
amide gel were directly sequenced using a 50-pL aliquot of the
double-stranded PCR products. A clonal band among a smear of
polyclonal products required further separation by denaturing gradient gel electrophoresis (DGGE).” To prepare products for DGGE,
a GC-clamped JHprimer (Table 1) was used in the second amplification step of the CDRIII assay. After DGGE and staining in ethidium
bromide, the band of interest was excised using a clean scalpel, and
DNA was extracted by freeze-thawing the gel slice three times using
a dry ice/ethanol slurry and a 37°C water bath. To prepare product
without the GC-clamped end for sequencing, 5 pL of a 1500 dilution
of the extract in water was reamplified using the JH nested and V670
primers, as described above. Excess primers, deoxynucleotides, and
buffer components were efficiently removed with Qiaquick-spin columns (Qiagen, Inc, Chatsworth, CA) according to themanufacturer’s
recommendations.
Sequencing reactions were performed in a Perkin Elmer-Cetus
Model 480 thermocycler (Perkin Elmer-Cetus, Foster City, CA) using one of the amplification primers, JH nested or VG7,,,along with
Tnq polymerase and four dye-labeled dideoxynucleotide terminators
supplied in the Taq DyeDeoxy Terminator Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA). Excess dideoxynucleotide
terminators were removed with a Centri-Sep column (Princeton Separations, Adelphia, NJ) before product analysis on an Applied Biosystems 373A automated DNA sequencer. Sequence data generated
with each primer were aligned (Sequence Navigator; Applied Biosystems) to ensure correct base assignment.
HODGKIN‘S DISEASE AND LARGEB-CELL
Primer
5 ’ 4 ‘ Sequence
Tubulin + strand
Tubulin - strand
AAG AAA TCC AAG CTG GAG l T C
G l T GGT CTG GAA TTC TGT CAG
CTG TCG ACA CGG CCG TGT A l T ACT G
ACC TGA GGA GAC GGT GAC C
ACC AGG GTC CCT TGG CCC CA
CGCCCGCCGCGCCCCGCGCCCGGC
CCG CCG CCC CCG CCC GAC CAG
GGT CCC TTG GCC CCA G
v870
JH
external
JHnested
JHnested (GC-
clamped)
CS
Case 1
Case 3
Case 8
TGG GTC ACA CGG GGG CAC GG
(replace Ve7d
GGA ATC AAT AAT CCC CGA AG
(replace JH nested)
TCC AAG TAC ATG AGA GTT GC
(redace JU nested)
include a 4-minute incubation in 1% (wt/vol) iodine in xylene to
remove the mercury introduced by the fixative.
High-molecular-weight ( H M W ) DNA was extracted from frozen
tissue as previously described?‘j The integrity of the isolated DNA
from both frozen and paraffin-embedded materials was evaluated in
a PCR-based assay using primers to the human beta tubulin gene?7
A 280-bp product, visualized on a 1.5% agarose gel (NuSieve 3:l
Agarose blend; FMC BioProducts, Rockland, ME) stained with 0.2
pg/mL ethidium bromide, confirmed the presence of amplifiable
DNA.
To prevent cross-contamination, either a newor an unused portion
of the microtome blade was used to cut the paraffin blocks. The
bath was cleaned, and the water was replaced before each sectioning.
No LPHD and corresponding LCL specimens were cut or extracted
on the same day. The DNA was physically extracted in an area free
of previous PCR products, and gloves were changed frequently during the procedures.
Complementarity Determining Region-Ill (CDR-Ill)
Clonality in Tumor Specimens
To assess the clonality of the LCL and LPHD specimens, a seminested PCR approach based on the technique of Wan et alz8 was
used. The primers used are listed in Table 1. A hot start procedure
was used to maximize the yield of specific pr0duct.2~Thirty PCR
cycles were used, with 40 seconds at 94°C to denature, 40 seconds
at 55°C to anneal, and 40 seconds at 72°C to extend the DNA. A
second round of amplification using 5 pL ofa 1:500 dilution of
the initial reaction products was performed under similar reaction
conditions with the JH nested primer replacing the initial JH primer
(sequence in Table I), but only 20 cycles were used.
The amplification products were analyzed in 8% nondenaturing
polyacrylamide gels (Ambresco, Solon, OH) in 1X TBE buffer
(0.089 m o m Tris-borate, 0.002 m o m EDTA pH 8.0). The positive
control consisted of 1:lO and 1:100 dilutions of Namalwa DNA (a
Burkitt’s lymphoma cell line known to have a rearranged IgH gene)
in germline DNA.w The negative controls consisted of either germline or polyclonal DNA from the myeloid leukemia cell line K562
or peripheral blood mononuclear cells (PBMC), respectively, and
sterile water.
Design of CS Oligonucleotide Primers and Probes
Computer analysis of the sequence was performed using the sequence analysis software package of the University of Wisconsin
Genetics Computer Group, Release 7.3. The sequences obtained
were compared with an index of previously published Dand JH
genes provided by Dr Harry Schroeder, Jr (University of Alabama,
Birmingham, AL). CS oligonucleotide primers and probes were designed with the help of a primer analysis program (Oligo version
4.1; National Biosciences, Inc. Plymouth, MN).
C o n j m t i o n of the Specificity of the CS Oligonucleotide
Probes
To confirm the specificity of the CS probes, a dot blot assay was
performed. Clonal CDRIII products derived from the LCL specimens
were retrieved, as well as CDRJII products from peripheral blood
lymphocytes (PBL) and the Namalwa cell line. After running an
aliquot on an 8% polyacrylamide gel to confirm the presence of
analytes, 5 pL and 2 pL were denatured in 200 pL of 0.4 N NaOH
for 30 minutes. The DNA was blotted to a nylon filter using a dot
blot apparatus (S&S Minifold I; Schleicher & Schuell, Keene, NH)
and was ultraviolet (UV) crosslinked.
Ten picomoles of probe were radiolabeled with [y3*P]-adenosine
triphosphate (ATP) using T4 Polynucleotide Kinase (Life Technologies, Gaithersberg, MD). Hybridization with 5 pmol of the labeled
probe proceeded for 4 hours at 10°C below Tm, as calculated from
the formula Tm = 4 (G + C) + 2(A + T), with constant agitation
maintained by a hybridization oven (Model 310; Robbins Scientific,
Sunnyvale, CA). Filters were washed once in 2X saline sodium
citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) for 15 minutes
at room temperature and then washed for another 15 minutes in a
pre-warmed portion of the same solution in the hybridization oven at
the original hybridization temperature. Autoradiography commenced
overnight at -70°C in a light-proof cassette with intensifying
screens.
Sequencing the Clonal CDRIII Product
Determining the Sensitivity of the CS Assay
The PCR products from cases showing a clear monoclonal band
with no detectable polyclonal background products on the polyacryl-
TOevaluate the sensitivity of the CS amplifications, serial 10-fold
dilutions of the LCL DNA were made into canier DNA (K562 or
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231 4
WICKERT ET AL
Table 2. Clinical Findings
Localization
Case No.
1
2
3
4
Age
lyr)/Sex
5
6
52lM
7
8
51lF
25lF
33lM
9
LPHD
7OlF
39lM
47/M
45/F
211M
Occurrence
R femoral LN
L axillary L N
R axillary LN
Paraaortic LN
Axillary LN
Mesenteric LN
Abdominal LN
SC LN
R axillary LN
HL-60). Amplifications were performed as in the nested CDRIII
assay using consensus primers, but with a few modifications. First,
the cycle number was increased to 35 in both the initial and nested
amplification steps. Aliquots ( 5 pL) of a 1:200 dilution of the initial
products were used as template in the second step. Finally, either
the JH nested or VeT0primer was replaced by the CS primer in the
nested PCR (Table 1). In addition, amplification of the serial LCL
DNA dilutions was performed using only the consensus primers
followed by hybridization with the corresponding CS probes to determine the sensitivity of the CS probe without a specific primer amplification step. All products were run on 2% agarose gels, transferred
to nylon filters (Turboblotter; Schleicher & Schuell), and probed as
described above.
Detection of the LCL Clone in the Corresponding LPHD
Undiluted DNA samples extracted from the LPHD specimens
were analyzed using consensus and CS primers and were probed
with CS probes as described in the sensitivity validation assays. A
total DNA equivalent of 200,000 cells was assayed in six separate
reactions based on the estimation that a 1 cm X 1 cm X 5 pm tissue
section contains approximately 500,000 cells. Controls consisted of
the I: 100 and 1: 1,OOO dilutions of LCL DNA and PBMC DNA, a
1:100 dilution of Namalwa DNA, and sterile water. Only the consensus primers were used in cases 6 and 9, in which a CS primer was
not available. All PCR products were electrophoresed on 2% agarose
gels, transferred to nylon filters, and probed as described earlier.
LCL
Colon
Spleen
R axillary LN
Paraaortic LN
R femoral LN
Mesenteric LN
L and R cervical LN
Breast mass
R axillary LN
+Q6 mos)
+
+
+
+(269 mos)
+
+(209 mos)
+(6 mosl
+
tissue blocks, and the LCL occurred as a large macroscopic
nodule. In case 6, the LCL existed as a large nodule amid
typical LPHD, and this nodule was microdissected for molecular study.
Molecular Data
DNA integrity. DNA from 15 paraffin-embedded tissue
blocks was extracted, and its suitability for PCR analysis
was evaluated by amplification of a 280-bp region of beta
tubulin, a gene present inall nucleated cells. Successful
amplification of this target was achieved in 66% ( 1 0 of 15)
of the available materials (Table 3). DNA extracted from
the two frozen tissues was shown to be intact by the same
procedure. In cases 6 and 9, a clonal CDRIII rearrangement
was demonstrable by PCR even though the ,&tubulin gene
was not amplifiable, probably because the small size of the
CDRIII fragment made the assay less sensitive to DNA degradation.
Clonality of LPHD and LCL. Material representative of
LPHD was analyzed with consensus primers to DNA sequences flanking the CDRIII of the IgH genes (Table 3). Of
the eight cases that could be studied, an oligoclonal pattern
consisting ofmany bands was observed in approximately
RESULTS
Table 3. Molecular Analysis
Clinical and Pathologic Data
The ages of the nine patients ranged from 21 to 70 years
(median, 45 years), and the male-to-female ratio was 5:4.
The most common localization of LPHD was the axillary
lymph nodes (n = 4), with no LCL site predominating. In
four of the five patients with concurrent disease, both processes presented at the same anatomic site. Of the four patients with subsequent diagnoses of LCL, all presented with
LCLs at sites other than the initial LPHD. The time intervals
in this second group were 6, 26, 209, and 269 months. The
clinical data are presented in Table 2.
All cases of LPHD were classified as the nodular subtype,
and the L&H cells in all seven cases studied had a B-cell
phenotype (CD20+ witWwithout CD74+). The LCLs were
also of the B-cell type in the eight cases studied. In all but
one case, the LPHD and the LCL were taken from separate
Subsequent
(interval)
Simultaneous
LCL
LPHD
Case No.
Tubulin
CDAlll
1
+(W)
0
2
3
-
-
4
+
+
5
+(W)
0
0
0
6
+(f)
0
7
NA
NA
P
+
P
+
Abbreviations: +, amplification detected;
8
9
Tubulin
CDAlll
+
C
+
C
0
-
i
-
+
+
-
_x
C
-
+(f)
C*
-
C
-, noamplification detected; C, clonal (1 or 2 bands); 0, oligoclonal (many discrete bands);
P, polyclonal (smear of products); W, weak; f, frozen tissue used for
analysis; NA, not available.
* IgH clonally rearranged by Southern analysis.
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HODGKIN’S DISEASE AND LARGE B-CELL LYMPHOMA
2315
B
A
A
Fig 1. PCR amplification ofCDRlll in LPHDand
LCL. The top panel shows LPHD-derived products,
and the bottompanel shows the corresponding LCLderived products.Lane A, molecularsize marker
(bottom band is 100 bp, and others are consecutive
multiples of 100); lanes B through F, cases l , 3, 6, 9,
and 8. respectively; lane G, case 8 on DGGE gel (size
not relevant to marker). Arrows point to clonal
bands. Lanes B, C, and D in the top panel show an
oligoclonal pattern of bands, whereas lanes E and F
show a polyclonal smear.
60% (five of eight) of the cases. Two cases yielded a polyclonal pattern with the amplification products forming a
smear along the expected size range, and no particular sized
product predominated. In case 2, with negative @tubulin
PCR, no amplification products were demonstrated.
In the corresponding LCLs, clonality was demonstrated
in 56% (five of nine) of the cases, as shown in Fig 1. Of
these five cases, a monoclonal band alone was observed in
three. Two bands were amplified in case 9, and a single band
among a dense polyclonal smear was seen in case 8. DGGE
was used to resolve the complex banding patternand to
isolate the clonal products from cases 8 and 9 for further
study. Results from the other four LCL cases included one
case with an oligoclonal patternand three cases withno
amplification products. Southern analysis for IgH
rearrangement was performed on cases 5 and 8, and. in each
case, a clonal population was demonstrated. PCR analysis
of these two cases, however, demonstrated a clonal product
in case 8, whereas no amplification products were observed
in case 5.
Studies with CS primers and probes. The sequence of
CDRIII was obtained in five LCL samples, and CS primers
and probes were designed
(Table 4). The specificity of the
fiveCS oligonucleotide probeswasconfirmed by dotblot
analysis. In each case, a hybridizationsignal was detected
only in the area blotted with the corresponding patient CDRIII
products. No cross-hybridization with other patient- or PBLderived CDRIII products was observed, as shown in Fig 2.
B
C
E
D
C
D
E
F
F
G
I-I-1-1
The sensitivity of amplification assays usingbothCS
primers and probes consistently reached the 1: 10,000 dilution in all three patients (cases l , 3, and 8) studied, as shown
in Fig 3. The sensitivity of the CS probe to targets generated
by consensus primers was 1: 10,000 in two cases, and 10fold less ( 1 :1,000) in one.
Five cases of LPHD associated with LCL were suitable for
further analysis using CS primers and/or probes
(Table 5). Only
CS probing of the LPHD could be performed in two patients
because size constraints of the clonal CDRIII products made
it impossible to design good CS primers as well. In these two
patients, the LCL clone was not detected in the corresponding
LPHD tissue. However, CS amplification revealed the presence
of the LCL clone in the LPHD component in two of the three
remaining patients. In caSe 3, LCL and LPHD were present in
the same anatomic site, but in different tissue bloch, whereas
case 8 presented with LCL at a different site 6 months after
the initial LPHD diagnosis (Table 2). In all experiments, the
negativecontrols(PBMCDNA,
1:lOO NamalwaDNA,and
sterilewater)displayed nohybridizationwiththeCSprobe
(Figs 4 and 5). Table 5 summarizes the resultsof these experiments.
TheCDRIIIproductsamplifiedwith
CS primersfromthe
LPHDtissueofcayes
3 and 8 weredirectlysequenced,and
thesequenceswerecomparedwiththoseobtainedfromthe
corresponding LCLs (Table 6). The sequences obtained from
case 8 were identical. The sequence from the LPHD of case 3
contained one position that cannot be assigned definitively, while
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2316
WICKERT
Table 4. CDRlll Sequence Data
~
Case No.
~~
GGCCTACTACTC
6
JH Segment
NDN
TGGTACTTCGATCTC
GGGGGCACGGTAGTGACTCCCGT
1
TGGGTCACAC
3
~
V. Segment
****
ttgattcc
"rGGGACTAAAGACTCTActtcggggatta
TGGGAAAGA
**********
GACTAT
TGCGCGA
*x******
8
GTCTGCAAAGTlTACACTGTTACCAAAAgcaactctcatgtact
TGCGAGA
tggaCTCC
*X***.
9
T G C m
GA'TTAC
GGAAGCGTAGTGGCN
********
Letters in upper case bold indicate sequence used as primer;lower case bold, reversecomplement of sequence used as primer; underscore,
sequence used as probe; *, sequence with homology to published D-region genes.
Abbreviation: NDN, D segment with franking N sequence.
the rest of the sequence is identical
on the LCL.
to that obtained previously
DISCUSSION
The cases of LPHD in this series had the classical morphologywith a macronodular architecture. However, the
definition ofan LCL arising in LPHD is still somewhat
controversial. We do not consider microscopic nodules of
L&H cells as evidence of LCL, and all the LCLs in this
study were macroscopically identifiable tumors that were
clearly delineated from the LPHD areas. In all cases except
one, the LCL was in a separate tissue block from the LPHD
studied. In case 6 , the two entities occurred in the same
block but could be readily separated from each other. It is
noteworthy that the LCL clone was not detected in the LPHD
portion in this latter case, suggesting that cross-contamination is preventable even when the two components occur in
the same tissue block.
We chose to amplify the LCL components with consensus
primers to the FR3 (V6,,,)and FR4 (JH) of the VH genes. The
small product amplified with these primers was ideal when
working with DNA extracted from routinely fixed, paraffinembedded biopsy specimens. Indeed, two of our LCL cases
showed no amplification at 280 bp with the 0-tubulin primers
8
9
6
but gave clonal bands of approximately 100 bpwiththe
consensus V and J primers. In contrast, two other LCL cases
were successfully amplified for the P-tubulin target but exhibited no CDRIII amplification products. PCR methods for
the detection of B-cell clonality using the consensus primers
to amplify the CDRIII may be falsely negative for a variety
of reasons, and about 30% of Southern blot positive cases
do not generate clonal CDRIII bands3'
Our current data and data from our previous study" do not
demonstrate a dominant clonal B-cell population in LPHD.
Multiple-sized CDRIII products with no clearly dominant
band were identified in all seven of our cases with amplifiable DNA. In contrast, analysis of the corresponding LCL
component showed clonal B-cell populations in six cases
(one case by Southern analysis only), whereas a pattern more
consistent with an oligoclonal proliferation was observed in
one other case. Of the six clonal cases, five were suitable
for analysis with CS methods.
The application of DGGE to purify clonal products from
a polyclonal background before sequencing was useful. A
discrete clonal bandwithout the heavypolyclonalbackground smear was obtained in case 8 after DGGE. Two
presumably clonal CDRIII bands were detected by routine
polyacrylamide gel electrophoresis in case 9. Whenthey
3
1
P
9
6
3
l
Fig 2. Specificity of hybridizationwith theCS probe. Replicatestrips were dot-blotted in duplicate, with theCDRlll productsamplifiedfrom
the cases indicated along the top of the figure (P,CDRlll products derived from PBMC of a normal donor). The CS probe used for hybridization
is indicated on the left of the figure. Hybridizationwas only demonstrated when the probe was applied to the products from the same patient.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
HODGKIN'S
B-CELL
DISEASE AND LARGE
LYMPHOMA
2317
A
panel shows an ethidium bromide-stained2% agarose gel electrophoresis ofCS products from case 8,
and the bottom panel showsthe resulting autoradiograph after Southern transfer and
hybridization with
labeled CS probe.Lane A, molecularsizemarker
(bottom band is 100 bp, and others are consecutive
multiples of 100): lane B, CDRlll products amplified
from LCL DNA (120,000targets); lanes C through G,
products obtained from serial 10-folddilutions of the
LCL DNA into HL-60 DNA; lane H, products from HL60 DNA alone; lane 1, water control. Specific CDRlll
products are
detectable down to the
dilution
(lane F).
B
A
C
B
D
E
C
F
G
D
H
E
I
F
G
N
I
A B C D E F G H I J K L M N
A
B C D E
F G H I J K
L
M
N
Fig 4. Detection of the LCL clone in LPHD (case 3). The top panel shows a 2% agarose gel of CDRlll products, and the bottom panel shows
the resuking autoradiograph after Southern transfer andhybridization with labeled CS probe. Lane A, molecular size marker(bottom band is
100 bp, and others are consecutive multiples of 100);lane B, 1:l.OOO dilution (120targets) of LCL DNA in HL-60 DNA; lane C, 1:lO.OOO LCL
dilution (12targets); lanes D and E, LPHD DNA amplified with CS primer; lanes F through K, LPHD DNA amplified with consensus primers
only; lane L, 1:lOO dilution of Namalwa cell line into K562; lane M, CDRlll amplified from PBMC isolated from a normal donor; lane N. water
control. CS products are identified in the LPHD tumor when either the CS primer or consensus primers are used for amplification.
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WICKERT ET AL
2318
A 0 C D E F G H I J K L M N
l
A
B
C
D
E
F
G
H
I
J
K
L
M
N
Fig 5. Detection of theLCL clone in LPHD (case8). The top panel showsa 2%agarose gel of CDRlll products, and the bottom panel shows
the resulting autoradiograph after Southern
transfer and hybridizationwith labeled CS probe. Lane A, molecular size marker (bottom band is
l00 bp, and others are consecutive multiples of 100); lane B, 1:1,000 dilution (120 targets) of LCL DNA in HL-60 DNA; lane C, 1:lO.OOO LCL
dilution (12 targets); lanes D and E, LPHD DNA amplified with CS primer; lanes F through K, LPHD DNA amplified with consensus primers
K562; lane M, CDRlll amplified from PBMC isolated from a normal donor; lane N. water
only; lane L, 1:lOO dilution of Namalwa cell line into
control. CS products are identified in the LCL and LPHD tumor when the CS primer is used, but are not detected in the LPHD DNA amplified
with only the consensus primers.
were independently excised, purified, and sequenced, the
resulting data were not interpretable. We then analyzed each
band using DGGE, and only the lower band proved to be
clonal andwas subsequently sequenced successfully. The
failure to produce good sequence data in the first experiments
was interpreted as contamination by irrelevant background
CDRIII sequences. The upper band in case 9, as visualized
in the standard gel, may have been a heterogenous mixture
of similar-sized CDRIII products.
The CS assays did not detect an occult clone in three of
the five LPHD tumors studied. Before concluding that the
clone is absent in the corresponding LPHD, several issues
need to be considered: ( 1 ) The IgH V region genes can
undergo somatic mutation, which takes place in germinal
center B cell^.'^^^^ The nodules in LPHD have the structure
of altered germinal centers, and it is possible that L&H cells
have a highrate of VH mutation, as do normalgerminal
center cells.3s Thus, if enough significant mutations have
occurred in the LCL clone, the specific oligonucleotides
based on its sequence may not be efficient in detecting the
original sequence present in the LPHD and may result in a
false negative. (2) Our sensitivity studies with serial dilutions
of tumor DNA revealed a consistent level of detection at
one tumor cell per 10.000 normal cells whenboth a CS
primer and a CS probe were used. Alteration or degradation
of cellular DNA in the archival tissue might have prevented
us from detecting tumor DNA at the single-copy level. To
increase the chance of detecting the neoplastic clone, a total
of about 200,000 cells were sampled in six assays per case.
(3) If the clonal population in LPHD was present only focally, it might not have been included in the tissue sample
studied. (4) The possibility of Tuq polymerase-induced er-
Table 5. Detection of theLCL Clone in the LPHD Tumor
Table 6. CDRlll Sequence Comparison
Case No.
Amplification Method
and Source
Case NO.
Specific Primer
Consensus Primer
-
LPHD
+
LCL
ND
% Homology
TGNGAAAGATTTTGGGACTAAAGACTCTA
II I I I I I I I I I I i I I I I I I I l l l l l l l l
TGGGAAAGATTTTGGGACTAAAGACTCTA
97 (28/29)
8
+
LPHD
ND
All results were confirmed by hybridization to a radiolabeled
probe: +, detection; -, no detection.
Abbreviation: ND, not done.
Sequence Between Primers
3
LCL
CS
TGCGAGAGTC TGCAAAGTlT ACACTGTTAC AAAA 100 (34134)
I I I I I I I I I I IIIIIIIIII IIIIIIIIII Ill1
TGCGAGAGTC TGCAAAGTlT ACACTGTTAC AAAA
Vertical lines indicate homology; underscore, probe sequence. Calculation of
the percentage of hoqology excluded the primer sequences (not included in the
figure). N indicates base assignment not possible.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
2319
HODGKIN’S DISEASE AND LARGE B-CELL LYMPHOMA
for clinical information, Karen Hansen for typing the manuscript,
and George Pallas for photography.
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