Acute Leukemias - Republican Scientific Medical Library

250 Chapter 20 · Minimal Residual Disease Studies in Acute Lymphoblastic Leukemia
20.2.2 PCR-based MRD Detection
PCR amplification of a specific DNA sequence or complementary
DNA (cDNA) unique to the leukemia clone
can permit identification of one malignant cell among
10 4 –10 6 normal cells, making it, in general, a slightly
more sensitive method of MRD detection than FC.
Two types of PCR targets can be used to detect MRD
in ALL patients: junctional regions of leukemia clonespecific
rearranged IgH and TCR genes; or leukemiaspecific
breakpoint fusion regions of chromosome rearrangements.
In addition to the commonly used targets
which are described in detail below, several studies have
suggested that the Wilms tumor suppressor gene, WT1,
aberrantly expressed in the majority of cases of AML
and ALL, may also serve as a useful target for MRD
analysis [9–21].
The deletion and random insertion of nucleotides
during IgH and TCR gene rearrangement generates
unique junctional sequences that can serve as clonespecific
markers of the leukemia that can be identified
at the time of diagnosis and used for serially MRD assessment.
The precise nucleotide sequence of the junctional
region can be used in the design of oligonucleotide
patient-specific primers for PCR amplification and
detection of MRD during and following treatment of the
leukemia [22]. The clone-specific IgH or TCR gene rearrangements
can be identified at diagnosis in 80–95% of
cases by using various PCR primer sets [22, 23]. Subsequently,
patient-specific primer and probe sets based on
the rearranged DNA sequence of the leukemic clone can
be generated and used for MRD detection.
Leukemia-specific (chromosomal) rearrangements
are also useful PCR targets for detecting MRD. Oligonucleotide
primers are designed at opposite ends of the
breakpoint fusion region so that the PCR product contains
the tumor-specific fusion sequences. In most of
the chromosome translocations common to adult ALL,
the breakpoints are spread over regions larger than
2 kb of DNA, which is the maximal distance that can
be reliably amplified [24]. Therefore, MRD detection
of the more common fusion genes, such as BCR-ABL resulting
from the t(9;22) and MLL-AF4 resulting from
the t(4;11), depends on identifying the resultant leukemia-specific
fusion mRNA. This fusion mRNA can be
used as a target for MRD analysis using PCR after the
fusion mRNA (consisting of transcribed coding exons)
is converted to cDNA using the enzyme reverse transcriptase
(RT). This technique is known as reverse tran-
scriptase PCR (RT-PCR). Two other fusion gene products
in ALL are amenable to MRD detection using RT-
PCR techniques. The E2A/PBX1 fusion gene resulting
from the t(1;19) translocation is found in approximately
5% of ALL cases, irrespective of age [25–33]. The TEL-
AML1 fusion gene product results from the cryptic
translocation, t(12;21) and occurs in as many as 25%
of children with precursor-B ALL [34–37]. Several studies
suggest that the results of MRD detection using RT-
PCR of TEL-AML1 are concordant with MRD detection
using PCR of IgH or TCR rearrangements [38, 39]. RT-
PCR of fusion genes is highly sensitive and specific. It
is also less labor intensive than PCR of clonal IgH or
TCR gene rearrangements since a single set of primers
can be utilized for each fusion gene product. Despite
these advantages, the general applicability of using leukemia-specific
fusion genes for MRD detection remains
relatively low, since only about one-third of both pediatric
and adult ALL cases harbor a recurring fusion gene
for PCR amplification.
Early PCR-based MRD studies used qualitative or, at
best, semiquantitative methods for detection of the leukemia-specific
target. These PCR methods relied on
endpoint measurements; that is, analysis of the reaction
product after PCR amplification is completed. MRD
measurements depended on multiple dilutions with
coamplification of standards and were cumbersome, error
prone, and technically demanding [40–42]. During
the last several years, real-time quantitative PCR (RQ-
PCR) has been introduced and has become the new
standard for PCR-based MRD analysis [38, 43–45] In
contrast to PCR endpoint quantification, RQ-PCR permits
accurate quantification during the exponential
phase of PCR amplification. This method has a very
large dynamic detection range over five orders of magnitude,
thereby eliminating the need for serial dilutions
of follow-up samples. In addition, the quantitative data
are quickly available since post-PCR processing is not
necessary. Therefore RQ-PCR is suitable for quantitative
detection of MRD using either junctional regions of IgH
or TCR gene rearrangements, or using breakpoint fusion
regions of chromosome aberrations.
PCR-based methods are very specific, highly sensitive,
and widely applicable to the majority of patients
with ALL. Recently, standardized methods for RQ-PCR
analysis have been published to provide more accurate
comparisons of MRD results from different laboratories
[24, 46]. However, the design of primers and probes for
detection of patient-specific IgH gene or TCR gene rear-