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From the Departments of Oncology and
*
Pathology,
The Johns Hopkins University School of Medicine, Baltimore, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR products using
capillary gel electrophoresis, which produces single nucleotide
resolution and provides improved diagnostic sensitivity over
conventional methods. However, with this marked increase in
resolution and sensitivity, it is necessary to re-define normal
variation of TCR- gene rearrangement in control tissues to allow
appropriate interpretation of monoclonality if present. Using DNA
capillary gel electrophoresis, we examined the spectrum
of normal patterns for TCR- in a variety of T-cell-rich,
histologically benign tissue types, including spleen,
lymph node, tonsil, and blood, and compared
this with the patterns in T cell lymphoma samples. We defined relative
peak heights as h1/h2, where
h1 represents the peak height of the largest peak above the
normally distributed population, and h2 represents
the peak height of the normally distributed curve. We found spikes in
almost 20% of histologically benign samples with relative peak heights
that were more than 0.5 and up to 1.5. We designated these as
pseudo-spikes, because they may be mistaken for monoclonal
spikes. In contrast, the relative peak height of the T cell
lymphoma samples that showed clonal rearrangement was much higher than
that of the pseudo-spikes, being at least 2 in 11/11 and at
least 3 in 10/11 cases. Our data suggest that peaks with relative
height of at least 3 represent a true clonal population in diagnostic
samples. Peaks with relative heights of less than 1.5 may be
insignificant, while peaks with relative heights between 1.5 to
3 may warrant further evaluation. Although capillary gel
electrophoresis is superior in assessing T cell clonality,
caution must be exercised when interpreting results, because
pseudo-spikes appear to be common in benign tissues with lymphoid
populations and are not necessarily indicative of clonal malignant T
cell population. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß- and a
small minority bearing the 
-heterodimer. During T cell
development, TCR- gene rearrangement occurs first, and only if
the rearrangement is not productive does rearrangement of ß
occur. Thus, almost all T cells have rearranged TCR- genes at the
molecular level even if the cell ultimately becomes
TCR-ß-bearing.3
The TCR- gene is also advantageous
for analysis because it has a relatively small number of somewhat
homologous V and J segments, making it simpler to analyze compared to
the other TCR genes.4
TCR- gene rearrangement is thus
the most commonly analyzed marker of T cell clonality. Historically, analysis of TCR gene rearrangement was done by Southern blotting, though it is now most commonly analyzed by polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE).5, 6, 7, 8, 9, 10 The presence of a distinct band of a single size indicates clonality, whereas diffuse staining spanning the expected PCR product size range indicates a heterogeneous collection of amplicons, consistent with polyclonality. However, several technical problems exist with these analyses. Standard-sized polyacrylamide gels lack single basepair resolution, and the distinction between single bands representing monoclonality and diffuse staining indicative of polyclonality can be subtle and interpretation can be somewhat subjective. Furthermore, standard PAGE lacks the resolution to detect oligoclonality or biclonality when the clones in question have rearranged gene products that differ in size by only a few basepairs. These patterns may appear erroneously as monoclonal spikes. PAGE is also not sensitive in detecting small populations of clonally rearranged cells in a polyclonal background. Finally, sizing of PCR products on PAGE is relatively imprecise, as it depends on rough extrapolation using adjacent size standards.
Capillary gel electrophoresis (CGE) with automated fluorescent fragment analysis has been used in the analysis of TCR gene rearrangement.11, 12 This technique produces single nucleotide resolution and allows objective quantification of the length distribution of PCR products present. As such, CGE may offer improved sensitivity in the analysis of TCR gene rearrangement and has potential advantage in being a quantitative or semiquantitative tool to detect small populations of clonal cells. It may be useful in the detection of minimal residual disease in T cell lymphomas or leukemias, and may also be a potential tool for early detection.
We assessed the sensitivity of CGE in detecting small amounts of T cell clones in a background of polyclonality and compared this to analysis by PAGE. As CGE has markedly increased sensitivity and nucleotide resolution, we found it necessary to redefine the spectrum of normality in various lymphoid tissues to allow appropriate interpretation of CGE-based TCR clonality assays. In several histologically benign tissues, we have found peaks that could be misinterpreted to indicate monoclonality. We have designated these as pseudo-spikes. By comparing the relative peak heights of pseudo-spikes in benign tissues to the true spikes found in known T cell lymphomas, we attempt to define a threshold to differentiate between non-neoplastic and neoplastic T cell populations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rearrangement in benign
peripheral blood samples, spleens, tonsils, and lymph nodes. The blood
samples were discarded specimens that had been submitted to the
laboratory for determination of Factor V Leiden status, from
individuals who have no clinical evidence of hematological malignancies
that would cause clonal rearrangement of their T cell receptor genes.
The other tissue specimens were obtained from paraffin blocks.
Pathological diagnoses were obtained from the hospital pathology
database, and the histology of the tissue blocks was reviewed and
confirmed by an independent hematopathologist (F. K. R.). The
spleens were histologically benign and had been removed for various
indications, including traumatic injury, congestive splenomegaly,
adenocarcinoma of the pancreas, and chronic pancreatitis. Tonsil tissue
was obtained from tonsillectomy specimens resected for tonsillitis. The
lymph nodes were obtained from axillary node dissections performed at
mastectomy for breast cancer and were uninvolved by tumor. In addition,
pathology specimens with histological diagnosis of T cell lymphoma were
analyzed to allow comparison to the benign samples. The diagnosis of T
cell lymphoma was based on the combination of morphology, flow
cytometry, and immunohistochemistry. Serial dilutions of DNA derived from a T cell line (Jurkat) and a normal population of T cells from a peripheral blood sample were mixed at various ratios to compare the limits of detection between conventional PAGE and CGE. The polyclonal T cells were enriched from peripheral blood nucleated cells by positive selection of CD2+ T cells with Dynabeads M-450 Pan-T (CD2, Dynal Inc., Lake Success, NY) and then eluted.
PCR
PCR was carried out essentially as described by Benhattar et al
with minor modifications.5
Briefly, a pair of consensus
primers was used (forward primer TV, 5'-AGGGTTGTGTTGGAATCAGG-3',
reverse primer TJ, 5'-CGTCGACAACAAGTGTTGTTCCAC-3') directed at the
3' end of the V segments and the 3' end of the J segments,
respectively. This primer pair detects 70 to 80% of TCR-
rearrangements and results in PCR products ranging in size from 160 to
190 bp. The forward primer was fluorescently labeled on the 5' end with
5-FAM (blue). The reaction was pseudo-nested; the first reaction was
carried out with only the reverse primer resulting in linear
amplification and the second reaction with both primer pairs, resulting
in exponential amplification. This procedure results in similar levels
of PCR amplification, increases specificity, and reduces the number of
false negative results.5
The first reaction was composed
of a total volume of 20 µl, comprised of 1x PCR buffer
(Perkin-Elmer, Foster City, CA), 0.2 µl 1% gelatin, 3 pmol reverse
primer, 2.5 nmol dNTP, 0.5 U AmpliTaq Gold (Perkin-Elmer), and 150 ng
DNA. Amplification was carried out in the Omnigene Hybaid thermocycler
using the following conditions: 94°C for 9 minutes, followed by 25
cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 73°C for
30 seconds. The second PCR reaction was standard bidirectional PCR,
performed with both TCR- primers. ß-Globin PCR amplification was
multiplexed into this reaction as an internal control to confirm the
presence of amplifiable DNA. A 20.6-µl volume of second reaction mix
was added to the 20 µl from the first reaction in the same tube,
making a total final volume of 40.6 µl. The second reaction volume
consisted of 1x PCR buffer, 0.2 µl 1% gelatin, 7.5 nmol dNTP, 20
pmol 5-FAM-labeled TCR- forward primer, 17 pmol TCR- reverse
primer, 7.5 pmol TET-labeled ß-globin forward primer (green,
5'-CAACTTCATCCACGTTCACC-3'), 7.5 pmol ß-globin reverse primer
(5'-GAAGAGCCAAGGACAGGTAC-3'), and 0.5 U AmpliTaq Gold. The forward and
reverse primers for both primer sets were at a 1:1 molar ratio in the
final volume, and the TCR- and ß-globin primers were at a 2.7:1
molar ratio. The ß-globin primer pair amplifies a 265-bp PCR product.
For the second PCR reaction, the samples were denatured at 94°C for 5
minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30
seconds, 73°C for 30 seconds, and completed with a 5-minute extension
at 73°C.
PAGE and CGE
The PCR products were analyzed both on PAGE with ethidium bromide
staining and by CGE with automated fluorescent fragment analysis on the
ABI 310 genetic analyzer (Perkin-Elmer). For the automated
fluorescent fragment analysis, 2 µl of the PCR products was mixed
with 12 µl deionized formamide and 0.5 µl of the internal size
standard GeneScan Tamara (red, Perkin-Elmer). After denaturation
for 5 minutes at 95°C, PCR products were size separated on the
genetic analyzer using GeneScan Performance Optimized Polymer 4C
(Perkin-Elmer) and analyzed, and size was determined by automated
fluorescence quantification using the GeneScan software. When the peak
height of amplified PCR product was off scale in the electropherogram
to the point where it could not be interpreted, the height limit
setting was re-adjusted accordingly on the GeneScan software. If the
peak height was still off scale despite the maximum setting allowed on
the software, the same PCR product was rerun using a shorter injection
time; failing this, less PCR product was loaded for a rerun.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rearrangement in 22 tonsils, 14
spleens, 9 lymph nodes, and 8 blood samples, all of which were
histologically benign. ß-Globin amplification was obtained for all
samples examined, confirming the presence of amplifiable DNA. All 53
samples showed polyclonal rearrangement on PAGE. When the samples were
analyzed using CGE, 50/53 samples showed a polyclonal distribution and
3/53 samples showed an oligoclonal distribution. We noted measurable
narrow peaks 1 to 2 bp in width above the polyclonal curve in 14/50
samples. We defined relative peak heights as
h1/h2, where
h1 represents the peak height of the largest
narrow peak above the normally distributed population and
h2 represents the peak height of the normally
distributed curve (Figure 1)
. When the spike is located at the edge of the polyclonal curve,
h2 is still defined as the peak height of the
normally distributed curve rather than the height of the polyclonal
curve at the position of the spike (h3) to avoid
a falsely elevated ratio (Figure 1B)
. A measurable
h1/h2 that is <0.5 was
noted in 4 tonsil samples but in none of the other three tissue types.
We considered peaks where
h1/h2 was <0.5 to
be noise (Figure 2A)
, and peaks larger than these to have possible significance.
Unexpectedly, peaks with
h1/h2 >0.5 were noted in
10/53 samples (19%), and were found across all four tissue types
analyzed. These included 6/14 (43%) spleens, 2/22 (9%) tonsils, 1/9
(11%) lymph nodes, and 1/8 (13%) blood samples. Because these
resembled the spikes seen in monoclonal populations, we designated
these as pseudo-spikes (Figure 2
, B-D). The relative peak heights
ranged from 0.56 to 1.37 (Table 1)
. Although pseudo-spikes were observed in all four lymphoid tissue
types studied, they were most prevalent among the spleen samples. The
most extreme pseudo-spikes were seen in two spleen samples, the first
from a patient with chronic pancreatitis (ratio 1.37) and the second
from a patient with carcinoma of the pancreas (ratio 1.28). For both
samples, DNA was re-isolated from the paraffin blocks and the analysis
repeated to exclude technical error. Similar pseudo-spikes with the
same size as previously recorded were found in both samples.
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rearrangement with CGE in 13
samples with a histological diagnosis of T cell lymphoma: 4 bone
marrow, 4 lymph node, 1 spleen, 1 colon, 1 testis, 1 lip, and 1 buccal
mass. All 13 samples amplified for the ß-globin control. Eleven of 13
samples demonstrated clonal TCR- rearrangement, 8 of which were
monoallelic and 3 biallelic. The relative peak height was at least 2 in
all 11 cases (Figure 3
and Table 2
). Two of the 13 samples showed a polyclonal TCR- gene rearrangement.
This is consistent with the limited sensitivity of the consensus primer
pair used in this study to detect all possible TCR- gene
rearrangements. Enhanced sensitivity may be achieved with additional
primer pairs or by combined TCR-ß and TCR- assays.13
When comparing PAGE and CGE in analyzing the T cell lymphoma cases, we
found concordance between PAGE and CGE in 9/13 cases (6 monoallelic, 2
biallelic, 1 polyclonal). In the 4 cases that were discordant, CGE
appears to provide more information than PAGE, showing monoclonal and
polyclonal rearrangement, respectively, for two samples for which no
bands were detectable on PAGE. For the remaining two samples, CGE
detected biallelic and monoallelic rearrangement, versus
monoallelic and polyclonal rearrangement on PAGE, respectively.
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. In contrast, when the same DNA samples were analyzed with PAGE, it
was not possible to be certain of the presence of monoclonal DNA even
in the 1:10 ratio sample (Figure 4A)
.
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,
E-G). In addition, we observed an oligoclonal pattern in 1/14 spleen,
1/9 lymph node, and 1/8 blood samples (Figure 2H)
. This finding is not
surprising, given the superior resolution provided by CGE, although its
clinical significance is uncertain.
We also analyzed 15 normal skin samples but failed to achieve
amplification for TCR- in any of the samples. This negative result
is possibly due to the scarcity of T cells in normal skin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pseudo-spikes are clearly common in benign lymphoid tissues when analyzed by CGE and are not indicative of a clonal malignant T cell population in diagnostic samples. They appear to be particularly common in spleen samples, being present in almost half of the samples analyzed. Caution needs to be exercised when interpreting TCR gene rearrangement results when using this as an indicator of clonality. We followed the clinical course of the patients with pseudo-spikes and found none to have developed a T cell malignancy in the intervening 1 year. More extensive follow-up may be warranted to determine the possible significance of such small peaks as a potential early detection marker.
We postulate that antigenic stimulation secondary to an underlying
pathology in some of the cases may account for the pseudo-spikes. The
immune system is stimulated in inflammatory conditions such as chronic
pancreatitis.14
Such antigenic stimulation may have
resulted in the preferential proliferation of one or more clones, which
manifested as a pseudo-spike. There are also studies showing
cell-mediated immunity to be stimulated in various
malignancies,15, 16
including specifically pancreatic
cancer17, 18
(Jaffee EM, Hruban RH, Biedrzycki B, Laheru
D, Schepers K, Sauter PR, Goemann M, Coleman J, Grochow L, Donehower
RC, Lillimoe KD, O’Reilly S, Abrams RA, Pardoll DM, Cameron JL, Yeo
CJ, manuscript submitted for publication). The majority of samples we
analyzed came from patients who had either infection or malignancy, and
this underlying process may have accounted for the pseudo-spikes
observed. Studying the pattern of TCR- rearrangement in tissues
harboring malignancy, chronic inflammation, or infection, particularly
those in which cellular immunity is activated, and comparing this with
the pattern of normal tissues from individuals without underlying
infection or malignancy may give further insight into the origin of
pseudo-spikes. Another theoretical explanation of the pseudo-spike
could be the random preferential amplification of one or more
rearranged TCR- gene in the early cycles during the PCR reaction
that is perpetuated in the subsequent cycles, resulting in a
pseudo-spike. However, we think this is unlikely, since the
pseudo-spikes were reproducible in independent experiments.
Can one define a threshold below which peaks can be explained away as pseudo-spikes and above which the test is indicative of a neoplasm? We were reassured to note that the spikes observed from known T cell lymphoma samples were more definitively clonal than the pseudo-spikes (ie, they had higher h1/h2 ratios). The relative peak height was >2 in all 11 positive cases and >3 in 10/11 positive cases. These ratios are much higher than what was observed with the benign samples, and may be a useful cutoff to distinguish between genuine monoclonal rearrangement and benign pseudo-spikes. Our data suggest that peaks with heights that are at least 3 times that of the normally distributed population is an appropriate threshold, indicative of a true clonal population in diagnostic samples. Such a cutoff correctly identified 10/11 known T cell lymphoma samples that were TCR-positive in our study. We have chosen 3 as a cutoff to be highly specific in diagnosing T cell clonality. This was a conservative choice based on our small sample size. Peaks that are less than 1.5 times that of the normally distributed population are seen in histologically benign samples, are likely to be insignificant, and may be disregarded as either noise or pseudo-spikes. Using these thresholds, all 53 histologically benign samples that were analyzed in our study would be correctly labeled as benign. Peaks that are from 1.5 to 3 times that of the normally distributed population should probably be designated indeterminate and warrant clinical correlation, further evaluation, and follow-up. More studies are required to determine the clinical relevance of these borderline peaks in the absence of other histological or molecular markers of T cell clonality. Additional studies with larger numbers of patients are warranted to confirm the appropriateness of the threshold values defined in this study.
Although assays for T cell clonality are used widely as adjuncts to diagnose malignancies originating from T cells, their use in the detection of minimal residual disease is not routine. This is because conventional PAGE lacks the sensitivity to detect small percentages of clones in a polyclonal background. Techniques such as Southern blotting or development of probes or primer sets unique to each patient’s monoclonal rearranged TCR gene have been used for detection of minimal residual disease.19, 20 The latter analyses require sequencing the clonal spike and designing a tumor-specific primer. Once it is designed, the patient can be monitored for the level of the tumor, with the potential of determining the presence of minimal residual disease and diagnosing molecular relapse.21, 22, 23 This may be particularly effective with the advent of real-time PCR technology that allows accurate and sensitive quantification.24 Though such analyses may seem somewhat daunting in current clinical laboratory settings, they may ultimately prove efficacious and cost-beneficial, since therapy for molecular relapse (where only small numbers of cancer cells have recurred) may be substantially more effective than therapy after morphological relapse occurs.
CGE is a useful technique for the monitoring of minimal residual disease given its superior resolution and sensitivity. As CGE provides fairly precise sizing of the rearranged clone, this information should probably be included in the clinical report. This would be helpful when evaluating for minimal residual disease, where a rearranged clone of the same size can be specifically sought. In this situation, it might be appropriate to use thresholds lower than what we have defined for diagnostic specimens. It may also potentially provide useful information regarding the amount of monoclonal cells present with respect to polyclonal T cells, although more studies are required to define thresholds for such information. Since monitoring of disease relapse typically depends on evaluation of blood or bone marrow samples, it is reassuring to note a relatively low frequency and heights of pseudo-spikes in the normal blood samples we have analyzed, suggesting that the distinction between normal and malignancy can be more clearly defined in these tissue types. We read with great interest the recently published paper by Sprouse et al.25 They describe an alternate method for approaching the same problem of quantifying in TCR-PCR assays the ratio of clonal to polyclonal components in samples containing both. Additional experiments may need to be performed to compare the two approaches directly.
CGE has better limit of detection and markedly better resolution than conventional PAGE, making it a valuable tool for diagnosing T cell neoplasms. However, one must account for the degree of noise and pseudo-spikes commonly observed in the histologically benign tissue samples. Once this is taken into account, CGE provides a markedly improved assay compared to conventional PAGE.
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Acknowledgments |
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Footnotes |
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Accepted for publication June 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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