JMD 2000, Vol. 2, No. 1
Copyright © 2000 American Society for Investigative Pathology & Association for Molecular Pathology
Detection of Clonally Restricted Immunoglobulin Heavy Chain Gene Rearrangements in Normal and Lesional Skin
Analysis of the B Cell Component of the Skin-Associated Lymphoid Tissue and Implications for the Molecular Diagnosis of Cutaneous B Cell Lymphomas
Minakshi Nihal,
Debra Mikkola and
Gary S. Wood
From the Department of Dermatology and the Skin Diseases Research Center, Case Western Reserve University; University Hospitals of Cleveland; and the Louis Stokes Cleveland VA Medical Center, Cleveland, Ohio
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Abstract
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A monoclonal B cell population is the hallmark of B cell neoplasms
including cutaneous B cell lymphomas (CBCLs). We modified and tested
several polymerase chain reaction (PCR)-based assays involving
amplification of immunoglobulin heavy chain (IgH) gene rearrangements
to optimize assays specifically for cutaneous lymphoid infiltrates. We
achieved greatest sensitivity with an assay employing IgH consensus
primers complementary to the framework 3 portion of the upstream
variable region and the downstream joining region. We studied 12
CBCLs, 6 nodal lymphomas and 7 cell lines. In 17/25 of these B
cell neoplasms (84%), we detected one or two dominant
bands, consistent with one or both IgH alleles being rearranged
in the neoplastic B cell clone. As expected, IgH PCR assays
produced diffuse smears in agarose gels or complex ladders in
polyacrylamide gels when polyclonal B cell controls (blood and tonsil)
were analyzed. However, in normal skin and non-CBCL skin
lesions, one or a small number of discrete bands were sometimes
detected. In certain cases, this made it difficult to
distinguish true positives (monoclonal CBCL) from false positives
(clonally restricted benign B cells). Correlation with
immunophenotyping confirmed that false positive results were confined
to samples with sparse or immunohistologically undetectable B cell
infiltrates. Pseudoclonal bands showed variable sizes in repeat PCR
reactions and could be distinguished from monoclonal bands by
polyacrylamide gel electrophoresis of pooled triplicate PCR products.
These findings suggest that molecular diagnosis using IgH PCR assays is
best suited for B-cell-rich infiltrates, and can be problematic
when applied to suspected T-cell-rich CBCLs, cutaneous T cell
lymphomas, or other lesions containing only few B cells unless
one is cognizant of the potential pitfalls. Furthermore, these
results demonstrate the presence of rare B cells in normal skin and
immunohistologically defined cutaneous T cell infiltrates. This
correlates with recent reports of sparse B cells within the lymph
draining from normal skin and may represent molecular evidence for a
trafficking B cell component of the skin-associated lymphoid tissue
(SALT). It also suggests a candidate B cell subset for the pathogenesis
of cutaneous lymphoid hyperplasia and CBCLs.
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Introduction
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Polymerase chain reaction (PCR)-based analysis of immunoglobulin heavy
chain (IgH) gene rearrangements is potentially useful for the analysis
of B cell clonality in CBCLs, other skin lesions, and noncutaneous
extranodal specimens exhibiting small size and/or sparse lymphoid
infiltrates. Such specimens are frequently not amenable to conventional
Southern blot analysis of genomic immunoglobulin gene rearrangements.
Nevertheless, the complexity of the IgH gene presents challenges to the
design of effective PCR-based IgH assays that are rapid,
straightforward, sensitive, and specific.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
The
variable region of the IgH gene is composed of a large number of gene
segments, each of which contains three hypervariable complementary
determining regions (CDR1, -2, and -3) and three relatively conserved
framework regions (FR1, -2, and -3). This structure is depicted in
Figure 1
. Using FR-directed oligonucleotide PCR primers reported to amplify IgH
gene rearrangements with sensitivity and specificity in lymphoid and
hematopoietic tissues, we developed and tested IgH PCR methods designed
for extranodal tissues such as the skin. We found that analysis of the
skin poses special challenges for the interpretation of B cell
clonality because sparse B cells trafficking through the skin can lead
to false positive results regarding the diagnosis of CBCL. However, by
adopting the strategies developed in this study, the clonality of most
cutaneous B cell infiltrates can be interpreted correctly. In addition,
our findings provide a context for future molecular biological
characterization of SALT B cells.
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Figure 1. Diagramatic representation of various regions of the immunoglobulin
heavy chain (IgH) gene.
The various framework regions
(FR) within the variable
region of the IgH gene are shown along with the positions of the
oligonucleotide primers used in the different PCR assays.
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Materials and Methods
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Tissue Specimens and Cell Lines
Twenty-five cases of B cell lymphoma (9 primary CBCL skin
specimens, 3 secondary CBCL skin specimens, 6 primary lymph node
specimens, and 7 neoplastic B cell lines) and 23 controls (4 reactive
tonsils, 3 normal bloods, 1 T cell lymphoma line (Jurkat), and 15
non-CBCL skin specimens including 1 lymphoid hyperplasia, 1 mycosis
fungoides erythroderma, 1 natural killer cell large cell lymphoma, and
12 normal skins) were used in this study. All human tissues were
obtained according to protocols approved by local institutional review
boards.
DNA Extraction
DNA was extracted from fresh-frozen tissue by proteolysis followed
by phenol-chloroform extraction.1
This involved overnight
incubation of minced tissues or cells with 50 µl of proteinase K (1
mg/ml) in a buffer containing 0.8x SSC, 0.2 mol/L NaCl, 0.5% sodium
dodecyl sulfate, and 1 mmol/L dithiothreitol at 37°C. This was
followed by phenol-chloroform extraction, precipitation of DNA with
95% cold ethanol, subsequent washing with 80% ethanol, vacuum drying,
and resuspension of DNA pellets overnight in 1 mmol/L Tris-HCl, 1
mmol/L NaCl, and 1 mmol/L EDTA.
FR1 IgH PCR
This method used a nested, multiplex PCR approach (see Table 1
). The first round used a mixture of the consensus primers from the
framework 1 region (HuVH 1/5, 2, 3, 4, and 6) and the joining region
primer LJH. The second round used the FR2A and VLJH primers to
amplify a dilution of the first-round products.2, 3, 4, 5
The
reaction was performed in a total volume of 100 µl and the final
concentrations of the reagents for the first round were 50 mmol/L KCl,
10 mmol/L Tris-Cl, pH 8.3, 200 µmol/L each dNTP, 2.5 mmol/L
MgCl2, 300 ng FR 1 primers (50 ng of each HuVH
primer), 75 ng LJH primer, 1 µg DNA template, and 2.5 U
Taq polymerase. Cycle conditions for round one were 95°C
for 15 seconds, 63°C for 30 seconds, and 72°C for 30 seconds for
the first 5 cycles and thereafter 35 cycles at 96°C for 15 seconds,
57°C for 30 seconds, and 72°C for 30 seconds. Round two of the PCR
used 50 mmol/L KCl, 10 mmol/L Tris-Cl pH 8.3, 200 µmol/L each dNTP,
1.5 mmol/L MgCl2, 200 ng FR2A primer, 400 ng VLJH
primer, 1/100 of first-round product, and 2.5 U Taq
polymerase. PCR conditions for this round were 95°C for 15 seconds,
63°C for 30 seconds, and 72°C for 30 seconds for 40 cycles. About 6
µl of PCR product was loaded onto an agarose TBE gel and bands were
visualized by ethidium bromide staining. Expected band size was
240–260 bp.
FR2 IgH PCR
This method used a seminested PCR approach involving a consensus
primer for the VH region and 2 consensus JH region primers (see Table 1
). The round first of PCR used the primer FR2A (a consensus VH primer
designed from homologous sequences in the FR 2 region) and the
LJH primer.6
The second round of PCR used FR2A and the
VLJH primer. VLJH is located just upstream to LJH. The components of
the PCR mixture in a total volume of 25 µl were 10 µmol/L
ß-mercaptoethanol, 67 mmol/L Tris-Cl pH 8.8, 16 mmol/L
(NH4)2SO4,
0.02% gelatin, 0.4 U Taq polymerase, 1 mmol/L each dNTP,
4.5 mmol/L MgCl2, 0.5 µg of each 5' and
3'primers, and 1 µg DNA template. The second round of the PCR also
used the same reaction mixture with the only differences in the
concentrations of a few reactants: 0.24 mmol/L each dNTP, 2.0 mmol/L
MgCl2, 0.05 µg of each 5' and 3' primer, and a
1/5000 dilution of first-round product. Cycle conditions used were
94°C for 5 minutes, followed by 2 minutes each at 94°C, 50°C, and
72°C for 40 rounds and another 20-minute extension at 72°C. PCR
product was analyzed on a 3% agarose TBE gel. Expected band size was
240–260 bp.
FR3 IgH PCR
This method involved a one-step PCR, using a universal VH primer
and a consensus JH primer (Table 1)
. The sequence of the VH primer is
based on the 3' end of the FR3 region.7
The final
concentrations of the reaction mixture were: 200 µmol/L each dNTP, 50
mmol/L KCl, 10 mmol/L Tris-Cl pH 9.0, 1.5 mmol/L
MgCl2, 100 µg/ml bovine serum albumin, 2.5 U
Taq polymerase, 0.45 µg (68 pmole) of each 5' and 3'
primer, and 2 µg DNA template in a total volume of 100 µl. PCR
consisted of 40 cycles of 1 minute each at 93°C, 56°C, and 73°C
and a 7-minute extension cycle at 73°C for complete extension and
annealing of PCR products. Seven microliters of PCR product were loaded
onto a 3% agarose TBE gel and run at 120 V for 2 hours. Bands were
visualized by ethidium bromide staining. Expected band size was 120
bp.
Radiolabeled Sequencing Gel Electrophoresis
To achieve greater band resolution, IgH PCR products were also
radiolabeled by 33P ATP kinasing and then
electrophoresed in 6% denaturing polyacrylamide gels. The upstream
primer was radiolabeled using T4 polynucleotide kinase (2 µl), 10x
kinase buffer (2 µl), and 40 µCi 33P ATP in a
total reaction volume of 20 µl. The PCR products were electrophoresed
at 60 W for 3 hours. The gel was vacuum-dried at 80°C and then
autoradiographed overnight at -70°C.
Immunoperoxidase Methods
Tissue specimens were snap-frozen in isopentane/dry ice, stored at
-70°C, cryostat-sectioned, and stained with a three-stage monoclonal
antibody/biotin/avidin-horseradish peroxidase immunolabeling method as
described previously.13
The monoclonal antibody panel
included CD3 (T cells), CD20 (B cells), and CD38 (plasma cells).
Controls included isotype-matched first stage antibodies of irrelevant
specificity and staining with various stages deleted.
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Results
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Technical Characteristics of the IgH PCR Assays
In initial experiments using positive control B cell lines and B
cell lymphoma tissue samples, the FR1 and FR3 assays yielded
reproducible results, whereas the FR2 assay produced inconsistent
results in terms of both the amount of PCR products and their band
patterns. Therefore, the FR2 assay was abandoned and only the other two
methods were used for the remainder of the study. The PCR products of
monoclonal B cell lines gave rise to one or two discrete bands when
electrophoresed in either agarose or sequencing gels. Resolution was
better in sequencing gels but agarose gels were generally adequate for
interpreting results (Figures 2
3
4
). In contrast, the PCR products of samples rich in polyclonal B cells,
such as tonsils, produced a diffuse smear in agarose gels and a dense
ladder of bands in sequencing gels. Analysis of samples lacking IgH
gene rearrangements, such as the Jurkat acute lymphoblastic leukemia T
cell line and the SCC-13 squamous cell carcinoma line, lacked any
detectable IgH PCR products.
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Figure 2. Agarose gels showing positive and negative results for B-cell clonality
in a variety of samples using three different IgH PCR assays whose
upstream primers are targeted to consensus sequences in the IgH gene
variable region known as FR1, FR2, and FR3. Lanes containing a dominant
clonal IgH gene rearrangement exhibit a discreet band
(lanes marked +), whereas
lanes containing only polyclonal IgH gene rearrangements show only a
diffuse smear (lanes marked
-). B, blank; P, positive control B cell lines;
C, carry-over negative control; J, Jurkat T cell line negative control;
T, tonsil polyclonal control; S, normal skin control; CBCL, cutaneous
B-cell lymphoma; NBCL, nodal B-cell lymphoma; M, marker.
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Figure 3. The impact of the number of PCR cycles on the detection of dominant
clonality. The B cell lymphoma cell line LTR228 was diluted in reactive
tonsil. The FR3 assay was used. Each lane contains 6 µl of
radiolabeled PCR product. The proportion of LTR228 DNA is listed as a
percentage above each lane. The three panels represent 30, 40, and 50
PCR cycles.
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Figure 4. Detection of restricted IgH gene rearrangements in normal skin.
Triplicate analyses of three normal skin samples
(A-C) are shown using the
FR1 assay. The lanes marked "mix" contain pooled PCR products from
the three preceding individual PCR reactions. Each lane contains 6 µl
of radiolabeled PCR product. Daudi is a positive control cell line
(one band). Tonsil and
blood are polyclonal B cell controls that show a complex smeared ladder
of many bands. CO is the carry-over lane that contains all reagents
except DNA. The pseudo-monoclonal band patterns seen in some lanes
(one or two bands) are
converted to a polyclonal complex band pattern in the lanes containing
the triplicate mixes.
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In titration experiments using 40 PCR cycles, the clonal detection
thresholds of the FR1 and FR3 assays differed from each other and also
varied with the proportion of polyclonal IgH gene rearrangements
present in the template DNA mixture. For example, when various
monoclonal B cell lines were diluted in SCC-13 DNA, which lacks any
polyclonal IgH gene rearrangements, the clonal detection threshold was
0.25 to 1% for the FR1 assay (Daudi, Raji, MC/CAR, and ST486 lines in
sequencing gels) and 0.1% for the FR3 assay (LTR228, MC/CAR, and ST486
lines in agarose gels). The particular B cell line used in these
studies had some effect on the clonal detection threshold, probably
because the consensus IgH primers do not amplify all IgH gene
rearrangements with equal efficiency.
In contrast, when the same experiments using 40 PCR cycles were
conducted with reactive tonsil DNA as diluent, the clonal detection
thresholds of the FR1 assay (Daudi, Raji, MC/CAR, and CA46 in agarose
gels) and the FR3 assay (LTR228, Ramos, and ST486 in sequencing gels)
were changed to the much less sensitive levels of 90 to 100% and 30 to
40%, respectively. LTR228 results are shown in Figure 3
. These
findings indicate that even small proportions of polyclonal B cells can
interfere with detection of the dominant B cell clone.
In support of this view, an intermediate threshold of 10 to 20% for
the 40-cycle FR3 assay in sequencing gels was obtained when the LTR228
and MC/CAR monoclonal B cell lines were titrated in 10% tonsil DNA
mixed with SCC-13 DNA. Similar titration experiments using the FR1
assay in agarose gels could achieve a sensitivity threshold of no
better than 90% for the Daudi, Raji, and MC/CAR lines. These findings
indicate that the FR3 assay has a significantly better sensitivity
threshold than the FR1 assay, probably because it is less affected by
background polyclonal IgH gene rearrangements.
Altering the number of PCR cycles also affected the clonal detection
threshold. For the LTR228 cell line diluted in tonsil, the greatest
sensitivity (10%) was achieved using 30 cycles. Representative
titration results are shown in Figure 3
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Sensitivity of the IgH PCR Assays
To determine the sensitivity of the FR1 and FR3 assays, 25 B cell
neoplasms were analyzed using both agarose and sequencing gels as
detailed in Table 2
. The same final result (dominant clonal versus polyclonal)
was obtained in most cases when analyzed by either agarose or
sequencing gel methods; however, band resolution was superior in
sequencing gels. This lead to a revised interpretations in some cases:
from FR1+ (agarose) to FR1- (sequencing) in two samples and from FR3-
(agarose) to FR3+ (sequencing) in five samples. Therefore, use of
sequencing gels is recommended because of their enhanced band
resolution. Based on the results of sequencing gels, there were 13 FR1+
samples (52%); all of these were also FR3+. There were 21 FR3+
samples, including eight FR1-/FR3+, yielding an overall
sensitivity of 84%.
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Table 2. Sensitivity of FR1 and FR3 IgH PCR Assays for Detecting Dominant B Cell
Clonality in 25 B Cell Neoplasms
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Specificity of the IgH PCR Assays
It was evident from the experiments described above that the FR1
and FR3 assays were able to distinguish B cell lymphoma samples
containing monoclonal IgH gene rearrangements from samples rich in
polyclonal IgH gene rearrangements or lacking such rearrangements
altogether. However, our focus was on methods suitable for recognizing
B cell lymphomas in extranodal sites such as the skin. Therefore, we
studied the band patterns produced by these assays in samples
containing a variable proportion of reactive B cells ranging from
absent or very sparse, as in clinically normal skin, to more abundant,
as in various non-CBCL lymphoid infiltrates. In sequencing gels, normal
skin showed either no IgH PCR products, a polyclonal ladder of bands,
or a clonally restricted pattern of one or a few bands. Non-CBCL
lymphoid infiltrates showed one of the two latter patterns. When the
PCR findings were correlated with tissue section immunoperoxidase
staining for B cell antigens, it was apparent that lymphoid infiltrates
containing small numbers of CD20+ B cells or CD38+ plasma cells were
the ones most likely to yield a clonally restricted IgH band pattern.
This included the normal skin specimens that sometimes contained rare
perivascular CD20+ or CD38+ B-lineage cells. In some instances the
restricted IgH band pattern contained only one or two bands and,
therefore, could be interpreted as a false positive result that could
be mistaken for evidence of a monoclonal B cell lymphoma.
Representative findings are shown in Figure 4
. Proof that this type of
false positive band pattern could be created by sparse polyclonal B
cells was obtained by diluting normal blood with the SCC-13 cell line.
As shown in Figure 5
, at titers of 1% or less lymphoid DNA, restricted IgH band patterns
emerged in sequencing gels and mimicked monoclonal IgH gene
rearrangements.
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Figure 5. Titration of normal blood in the squamous cell carcinoma cell line SCC
13. The pattern of IgH gene rearrangements using the FR1 assay is shown
at various concentrations of blood. Each lane has 6 µl of
radiolabeled PCR product. At lower percentages of blood corresponding
to sparse B cells, a pseudo-monoclonal band pattern emerges.
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When separate PCR products from multiple amplifications of the
same pseudoclonal specimen were electrophoresed side by side, it was
apparent that the band pattern varied. This was consistent with
amplification of different IgH gene rearrangements that randomly gained
an advantage in early cycles of different PCR reactions. When these
products were pooled and electrophoresed, they exhibited an additive
complex band pattern that contrasted with the truly monoclonal samples
that consistently produced the same clonal band pattern (Figure 4)
.
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Discussion
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In the current study, we have modified and thoroughly tested PCR
methods that afford improved resolution of discrete IgH gene
rearrangements and detect dominant clonality in approximately 84% of B
cell neoplasms including primary CBCLs. The detection of clonal IgH
gene rearrangements in all seven B cell lines tested suggests that the
failure to detect clonality in some biopsy specimens was due to
competition from background polyclonal B cells rather than failure of
the PCR primers to amplify the tumor-specific IgH gene rearrangements.
However, the latter possibility cannot be completely excluded. Although
separation of PCR products in agarose gels was adequate in most cases,
there were some instances where false negative or false positive
results were seen in agarose gels. Therefore, sequencing gels are
preferred. The enhanced resolution provided by sequencing gels is also
advantageous in certain other circumstances. These include documenting
the same clonal rearrangement in multiple specimens from the same
patient during lymphoma staging, monitoring therapeutic response or
detecting early relapse.
Although there were no FR1+/FR3- cases in our current series, it is
entirely possible that such cases will be encountered. Therefore, use
of both the FR1 and FR3 assays is advised to maximize the detection of
dominant B cell clones. Overall, the FR3 assay proved to have a more
sensitive clonal detection threshold ranging from about 0.1 to 30%
when the background content of polyclonal B cells ranged from about 0
to 50%, respectively. Although the titration studies performed using
neoplastic B cell lines provide a rough indication of the clonal
detection threshold of these PCR assays, results may differ for
individual B cell lymphomas because certain IgH gene rearrangements may
be amplified better than others. These IgH gene rearrangements may be
more complementary to the IgH consensus primers.
We have also shown that sparse reactive B cells in extranodal tissues
such as the skin can result in clonally restricted band patterns that
can appear as false positives mimicking the true positive pattern
produced by B cell neoplasms. This observation underscores the
importance of using negative controls relevant to the tissue of
interest. Whereas reactive tonsils or lymph nodes are useful polyclonal
controls for nodal lymphomas, normal skin and inflammatory dermatoses
are more appropriate polyclonal controls for cutaneous lymphomas. This
important point has not been highlighted in prior studies of B cell
clonality employing PCR methods to detect IgH gene
rearrangements.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15
The false-positive pitfall inherent in analysis of skin lesions can be
avoided by an algorithmic approach that combines immunopathology with
gene rearrangement analysis (Figure 6)
. If immunopathological analysis demonstrates many B cells or plasma
cells, then IgH PCR is unlikely to produce a false positive pattern due
to restricted IgH gene rearrangements arising from sparse B cells. In
tissue samples with sparse B-lineage cells, IgH PCR analysis must be
interpreted with great caution. Based on our findings that showed
emergence of a pseudoclonal IgH band pattern at a titer of about 0.5%
polyclonal B cells (see Figure 5
), we set this limit conservatively at
about 5%. Without other supportive findings, a monoclonal pattern
observed in this setting should not be used as evidence for B cell
neoplasia. In these cases, electrophoresis of pooled products from
three or more PCR amplifications of the same sample can be helpful
because the random pseudoclonal band patterns will be additive creating
a complex band pattern while the true clonal band pattern will be
consistent (see Figure 4
). This has particular relevance for diagnosing
so-called "T-cell-rich B cell lymphomas" including lymphomatoid
granulomatosis. It is also relevant to the question of how frequently
IgH gene rearrangements occur in unequivocal T cell lymphomas and
leukemias, samples of which might contain sparse non-neoplastic B
cells. Although this study has focused on the skin, it is apparent that
a similar approach is required for the analysis of B cell clonality in
other extranodal sites that normally contain few or no B cells.
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Figure 6. Combined immunopathological/molecular biological algorithm for the
analysis of IgH gene rearrangements in extranodal tissues such as the
skin. This approach should help avoid false positive results secondary
to sparse B cells.
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Although it is possible that circulating B cells trapped in the blood
vessels of biopsy specimens may have contributed to the clonally
restricted B cells detected in some samples of normal skin, they were
unlikely to be the sole explanation for this phenomenon because some
normal skin samples lacked any detectable IgH PCR products whatsoever.
We believe that this clonally restricted B cell pattern is molecular
evidence for a B cell component of the skin-associated lymphoid tissue
(SALT).16
Presumably, these B cells traffic from the blood
vessels to the lymphatics via the extracellular dermal compartment and
thus could be present in each of these microenvironments at the time of
skin biopsy. This view is consistent with our immunopathological
studies demonstrating occasional extravascular B cells in the dermis of
normal skin and with prior reports showing that 3 to 5% of
tumor-infiltrating lymphocytes in primary cutaneous melanomas and
squamous cell carcinomas, as well as in cutaneous metastases, are CD20+
B cells.17
It is also supported by recent studies showing
that approximately 0.5% of the lymphoid cells in the lymph draining
normal human skin are, in fact, B cells.18
These
observations are exciting because they identify the IgH gene
rearrangements of a B cell subset that is the most likely candidate for
the origin of cutaneous B cell lymphoid hyperplasia and CBCLs. Future
studies aimed at the isolation and characterization of these putative
SALT B cells may help to elucidate the pathogenesis of cutaneous B cell
lymphoproliferative disorders.
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Footnotes
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Address reprint requests to Gary S. Wood, M.D., Chief, Dermatology Service 11-G(W), Louis Stokes Cleveland VA Medical Center, 10701 East Blvd., Cleveland, OH 44106.
Supported by a grant from the Dermatology Foundation, National
Institutes of Health grants P30-AR39750 and R21-CA75201, and Merit
Review funding from the Medical Research Service of the Department of
Veterans Affairs.
Accepted for publication October 19, 1999.
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Top
Abstract
Introduction
