(American Journal of Pathology. 1999;154:7-9.)
© 1999 American Society for Investigative Pathology
Advances in Molecular Hematopathology
T-Cell Receptor 
and bcl-2 Genes
Timothy C. Greiner
From the University of Nebraska Medical Center, Omaha, Nebraska
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Introduction
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The two articles by Signoretti et al1
and
Bohling et al2
published in this issue of the
Journal demonstrate ongoing advances in amplifying T-cell
receptor 
gene rearrangements (TCRGR) and the translocation of
the major breakpoint region of bcl-2 with the immunoglobulin heavy
chain gene (MBR/JH). Both use variations in polymerase chain reaction
(PCR) technology. During the first few years' use of PCR in these two
genes, analysis of PCR products occurred in standard agarose and
polyacrylamide gels. There have been many technical advances since the
1988 description of manual PCR to detect MBR/JH rearrangements by
Stetler-Stevenson et al.3
Recent progress has seen the
utilization of fluorescent detection and methods of separating gene
rearrangements focused on the differences in DNA sequences of the
rearrangements. Three key methods are single-strand conformation
polymorphism (SSCP) analysis,4
denaturing gradient
gel electrophoresis (DGGE),5
and temperature gradient gel
electrophoresis (TGGE).6
Pathologists interested in
understanding the basics of DNA sequence separation technologies should
read these three articles.
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Principles of Analyzing T-cell Receptor Gene Rearrangements
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One important feature facilitating the amplification of the T-cell
receptor (TCR) gene is the limited number of variable region and
joining region gene segments that have been described in tumors. These
include four families of variable segments, V1–8 (1–8 are closely
related in sequence), V9, V10, and V11, with three groups of
joining segments, J1–2, JP, and JP1–2.7-9
The
total number of gene segments is much lower than in the T-cell receptor
ß or the immunoglobulin heavy chain genes and there are also no
diversity gene segments.7-9
Therefore, it is easy for
molecular pathologists to design primers to cover rearrangements of all
of the variable region and joining region segments of the TCR gene.
Each of these families or groups of segments requires a unique primer
because there is insufficient homology to use just one consensus primer
for the variable region and one primer for the joining region
genes.
A review of the literature reveals that there are two main groups of
protocols: those which detail the amplification of all gene segments
and those which amplify only the most common gene segments used in
TCRGR. The most frequently used variable region and joining region
segments in peripheral T cell lymphomas are the Group I variable region
(V1–8) at 80% and the J1/J2 group at 60%.9
Thus, common primers can detect approximately 60–80% of TCRGR
according to the frequency of TCR gene segments used as described by in
Theodorou et al.9
V9, V10, and V11 are used in
decreasing order, and JP and JP1/JP2 are used in less than
20% of TCRGR.9
The detection frequency of TCRGR with
common primers in patient series tends to be higher than this due to
the natural tendency to build a collection from positive index cases.
It is also higher because biallelic rearrangements usually consist of
one rearrangement composed of the commonly used segments and another
composed of one of the infrequently used gene segments. Protocols using
only a limited number of primers will fail to detect the TCRGR with
the infrequently used genes. To test a protocol for detecting
rearrangements with JP1/2, try amplifying DNA from MOLT4 cells.
In rearrangements of the immunoglobulin gene, maximum detection
capabilities with Framework III primers are limited to about 80% due
to a combination of mutations at primer sites and the presence of
unknown gene segments. Unlike the immunoglobulin gene, we can expect to
detect 90–100% of Southern blot-confirmed TCRGR when
primers to all TCR gene segments are used.10
Therefore,
I think pathologists are obligated to use all of the known TCR
genetic information in designing protocols for their laboratory,
regardless of whether SSCP, DGGE, TGGE, sequencing gels, heteroduplex,
or high density polyacrylamide gels are used. To do less than that
would, I believe, unnecessarily risk missing a TCRGR and may affect
patient care.
The use of precast gels in thermally controlled SSCP (Signoretti
et al1
in this issue) to analyze T-cell receptor gene
rearrangements is a good addition to recent descriptions of SSCP in
various T-cell receptor gene families including 
, ß, and
genes. Certainly the use of the precast gels, as also described by Kaul
et al11
using a PhastSystem (Pharmacia, Uppsala,
Sweden), may be faster than pouring gels for either DGGE or SSCP
systems. The goal of controlling temperature during gel electrophoresis
is fundamentally important in any method for separating DNA by sequence
to obtain high resolution of the DNA bands, as in SSCP. The use of SYBR
Green II (Molecular Probes, Eugene, OR) appears to be a useful
alternative to silver staining of single-stranded DNA in SSCP.
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Alternative Gel Systems for TCRGR
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Many methods have been described, ranging from early agarose
gel-based electrophoresis, which separates DNA by length, to a variety
of systems that separate DNA by sequences as well as length. A highly
robust analytical electrophoresis system is desirable. The minimum
standard is high density polyacrylamide gel electrophoresis to obtain
sufficient discrimination between the amplicon products, which do not
vary greatly in length with TCR due to the lack of a diversity
region and limited insertions at the junction or N region.
Electrophoresis assays based on DNA sequence separation are more robust
in separating DNA, especially biallelic TCRGR of the same
length.10
These are also useful in establishing with
certainty that residual disease of a clonal neoplasm is present, based
on a comigration in the gel of the rearranged band. The performance of
sequence separation systems in mutation detection are nearly
equivalent, yet remain dependent on specific DNA sequence and
institutional expertise. Separation of TCRGRs in DGGE is maximized
by the incorporation of a guanine-cytosine (GC) clamp in the PCR
product.10,12
Representative reports suggest the use of
TGGE in TCRGR13-15
appears equivalent to results seen
in DGGE and SSCP. Newer methods involving fluorescent labeling of
TCR ß16
and 17,18
GR PCR products with,
eg, the ABI Gene Scan system (Applied Biosystems, Foster City,
CA) a type of fluorescent SSCP, have been described; however,
the instrumentation is very expensive. I and others have observed that
this system discriminates very well by length16-18
and it
has the added potential advantage of calculating comparisons
between peak heights. This may prove useful in resolving a difficulty
in all systems: discriminating oligoclonal expansions from neoplastic
clones.
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Fluorescent Detection System
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The article by Bohling et al2
describes a new way to
detect PCR products that may lend itself to real time analyses, an
elusive goal of several companies over the last five years. A recent
advance in analyzing fluorescent- labeled MBR/JH products included the
application of the ABI Gene Scan (ABI, Molecular Probes) in a standard
polyacrylamide gel.19
This system still represented a
post-PCR electrophoresis. The fluorescent detection with SYBR Green
I in the glass tube as described by Bohling et al,2
however, uses properties of change in fluorescence that occurs with DNA
strand association/disassociation coupled with measuring the
temperature at which the DNA denatures. Such innovations are to be
encouraged as laboratories continue changing from standard analyses of
PCR products in labor-intensive agarose and polyacrylamide gels.
Background references that readers would find useful in
understanding the technology described in this manuscript include the
schematic article on the LightCycler instrument by Wittwer et
al20
and the principles of measuring DNA melting curves
described by Ririe et al in 1997.21
These two articles
break new ground in showing a potential application combining an
understanding of DNA melting characteristics and fluorescent detection
technology.
The application of this technology appears best suited for the
detection of abnormal events such as neoplastic translocations,
infectious disease agents, or mutations that have a significant
difference in melting temperature. Members of Wittwer's group have
previously illustrated a possible use in infectious diseases by
amplifying the hepatitis B surface antigen21
with the
LightCycler and others have demonstrated the capability in identifying
Leptospira species.22
Most recently a
LightCycler protocol has been published to identify a hemachromatosis
gene mutation using yet another advance by incorporating the use of
peptide nucleic acid probes23
during PCR. Since amplified
polyclonal JH or TCR rearrangements would affect fluorescent
changes, it is not likely that this technique could be adapted easily
in these areas.
The possibilities of incorporating this detection technology may be
limited by the practice of confirming the identity of PCR products by
hybridization techniques, such as dot blots or ELISA-based technology,
along with the trend of directly sequencing PCR products. One concern
about relying on DNA melting profiles to identify a specific product is
that another DNA sequence may have the same melting temperature. A
thorough understanding of DNA melting technology should, however, allow
for the development of specific applications, such as the MBR/JH
t(14;18) translocation, provided there are no known nonspecific
products such as cross-reaction with Epstein-Barr virus.24
One slight drawback with previous generations of rapid cyclers, which
we have experienced in our laboratory, is difficulty in loading and
handling the capillary tubes. The innovators have attempted to address
this with a new plastic loading unit for the capillary tubes that may
make it more user friendly.2
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The Problem of Sporadic bcl-2 MBR/JH Rearrangements
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MBR/JH rearrangements have been described in individuals with no
disease or with reactive hyperplasia.25,26
The best way to
identify sporadic bcl-2 translocations is by duplicate tube analysis
and they can usually be avoided by using a nonnested
protocol.27
If the suspect translocation is not repeatable
in multiple tube assays it is best regarded as sporadic when one is
considering a primary diagnosis. In follow-up biopsy specimens in an
established patient the best course of action is to use comparative
analysis for comigration of MBR/JH in the two biopsy specimens.
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Future of Molecular Hematopathology
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Advances during the past decade have resulted in numerous
protocols for detecting gene rearrangements and translocations in
lymphoma and leukemia by PCR. Therefore, gel systems and primers vary
from institution to institution, making comparisons difficult. The
Association for Molecular Pathology and the National Committee on
Clinical Laboratory Standards are making ongoing efforts to determine
whether consensus protocols can be devised.28
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Footnotes
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Address reprint requests to Timothy Greiner, M.D., Associate Professor, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135. E-mail: tgreiner{at}mail.unmc.edu
Accepted for publication November 16, 1998.
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References
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Signoretti S, Murphy M, Cangi MG, Puddu P, Kadin ME, Loda M: Detection of clonal T-cell receptor gene rearrangements in paraffin embedded tissue by PCR and non-radioactive SSCP analysis. Am J Pathol 1999, 154:67-75[Abstract/Free Full Text]
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Bohling SB, King TC, Wittwer CT, Elenitoba-Johnson KSJ: Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis. Am J Pathol 1999, 154:97-103[Abstract/Free Full Text]
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Stetler-Stevenson M, Raffeld M, Cohen P, Cossman J: Detection of occult follicular lymphoma by specific DNA amplification. Blood 1988, 72:1822-1825[Abstract/Free Full Text]
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Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T: Detection of polymorphisms of human DNA by gel electrophoresis as single-stranded conformation polymorphisms. Proc Natl Acad Sci USA 1989, 86:2766-2770[Abstract/Free Full Text]
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Fischer SG, Lerman LS: DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci USA 1983, 80:1579-1583[Abstract/Free Full Text]
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Ke SH, Wartell RM: Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA: determination by temperature-gradient gel electrophoresis. Nucleic Acids Res 1993, 21:5137-5143[Abstract/Free Full Text]
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LeFranc MP, Forster A, Baer R, Stinson MA, Rabbitts TH: New subgroups in the human T cell rearranging genes: nine germline variable genes belonging to two subgroups. Cell 1986, 45:237-246[Medline]
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Huck S, LeFranc MP: Rearrangements to the JP1, JP and JP2 segments in the human T-cell rearranging gene (TRG) locus. FEBS Lett 1987, 224:291-296[Medline]
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Theodorou I, Raphaël M, Bigorgne C, Fourcade C, Lahet C, Cochet G, LeFranc MP, Gaulard P, Farcet JP: Recombination pattern of the TCR locus in human peripheral T-cell lymphomas. J Pathol 1994, 174:233-242[Medline]
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Greiner TC, Raffeld M, Lutz C, Dick F, Jaffe ES: Analysis of T cell receptor- gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products: correlation with tumor-specific sequences. Am J Pathol 1995, 146:46-55[Abstract]
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Greiner TC: Analysis of TCR gene rearrangement by two methods: Denaturing gradient gel electrophoresis and laser-scanning of fluorescent-labelled products. Oksenberg JR eds. The Antigen T Cell Receptor: Selected Protocols and Applications. 1997, :pp 407-431 R.G. Landes Company, Austin, TX,
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Simon M, Kind P, Kaudewitz P, Krokowski M, Graf A, Prinz J, Puchta U, Medeiros LJ, Sander CA: Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor -chain gene rearrangements in lymphoproliferative diseases. Am J Pathol 1998, 152:29-33[Abstract]
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Luthra R, McBride JA, Hai S, Cabanillas F, Pugh WC: The application of fluorescence-based PCR and PCR-SSCP to monitor the clonal relationship of cells bearing the t(14;18)(q32;q21) in sequential biopsy specimens from patients with follicle center cell lymphoma. Diagn Mol Pathol 1997, 6:71-77[Medline]
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Introduction
Principles of Analyzing T-cell...
