- What is a BCR-ABL1 monitoring test?
A monitoring test quantitatively measures the blood level of BCR-ABL1 fusion
transcript during the course of TKI treatment. This transcript is a marker for
the presence and amount of transcriptionally active Philadelphia-chromosome
positive leukemia cells in chronic myeloid leukemia patients.
(Baccarani et al., J Clin Oncol 2009). Total RNA is in general extracted
and purified from leukocytes, then reverse transcribed (RT) and the cDNA product
is quantified by fluorescent real-time quantitative polymerase chain reaction
(qPCR). The RT-qPCR test must also detect an endogenous control transcript (EC)
to assess the quality and quantity of RNA and to normalize for potential
differences between tests. Therefore, the result of this test is expressed as a
percent ratio between the BCR-ABL1 and EC copy number (100 x BCR-ABL1/EC).
- How are BCR-ABL1 monitoring results reported?
Monitoring of response to TKI and results interpretation are based on the
variation of BCR-ABL1 expression levels over time. These variations are measured
in fold change or more commonly in log (base 10) change. For example, a change
in percent ratio (100 x BCR-ABL1/EC) from 50% to 0.5% corresponds to a 100-fold
decrease or a 2-log decrease (log(50)-log(0.5)=log(50/0.5)=log(100)=2). A 3-log
decrease is equivalent to a 1000-fold decrease; a 3.16-fold increase is
equivalent to a 0.5-log increase. In most cases, clinically relevant changes in
BCR–ABL expression levels are at least 3- to 10-fold and are confirmed by two
subsequent measurements within a period of a few months before any change in
therapeutic strategy. (Baccarani et al.,
J Clin Oncol 2009).
- What is the International Scale for standardization of quantitative BCR-ABL1
result reporting?
The International Scale (IS) was established in 2005 to standardize quantitative
BCR-ABL1 measurements across tests and laboratories.
(Hughes et al., Blood 2006). The IS is anchored to the baseline BCR-ABL1
expression level from the International Randomized Study of Interferon vs STI571
(IRIS) trial (100% IS) with a major molecular response (MMR) corresponding to
0.1% IS. (Hughes et al., N Engl J Med
2003). The IRIS trial and follow up studies have demonstrated that achieving
MMR, or a 3-log reduction in BCR-ABL1 expression from the standardized baseline
level, is a key clinical outcome. Percent ratios on the IS are obtained by
multiplying the percent ratio (100 x BCR-ABL1 / EC) determined at local labs by a
test-specific conversion factor (IS % ratio = local % ratio x conversion
factor).
- What is the importance of the IS?
Monitoring tests that quantitatively measure the blood level of BCR-ABL1 fusion
transcript use a variety of commercial and laboratory-developed reagents and processes.
This includes different RNA preparation methods, real-time PCR instruments, real-time
technologies, enzymes, fluorophores, quenchers, and standard curve materials. As such,
reported BCR-ABL1 measurements vary greatly between laboratories. The importance of the
International Scale (IS) is that it standardizes quantitative BCR-ABL1 measurements across
tests and laboratories, facilitating interlaboratory studies, patient portability, and a
harmonized definition of treatment response.
- How do I standardize my lab to the IS?
The consensus meeting in October 2005 at the NIH in Bethesda proposed the
preliminary guidelines for establishment of an international scale (IS) that
could be applied at individual centers. The process for the local laboratory to
convert its in-house results to the IS involves i) adoption of the consensus
principles established by the Bethesda group; ii) testing a set of reference
standards to establish a laboratory-specific conversion factor; and iii)
multiplying all local BCR-ABL1 values by the conversion factor to express the
results according to the IS. (Hughes et
al., Blood 2006; Branford et al.,
Blood 2008). To test the accuracy of this approach, a set of clinical
samples assayed in the local laboratory would then need to be sent to an
international reference laboratory to verify that the conversion factor provided
an accurate conversion. Currently, and until reference standards become broadly
available, patient specimens must be exchanged between the local laboratory and
an IS Reference Laboratory to establish a laboratory-specific conversion factor.
- Can any lab become standardized to the IS?
Any local laboratory can exchange patient samples with an IS Reference
Laboratory to align its assay to the IS. However, the laboratory should first
ensure that it meets specific pre-analytical and analytical criteria prior to
implementation into clinical practice. Some critical parameters for quantitative
measurement of BCR-ABL1 and reporting on the IS include minimum number of
leukocytes processed per RNA extraction, endogenous control gene identity and
minimal copy number detected per reaction, total assay precision and accuracy,
linearity, and limit of quantification for BCR-ABL1.
(Branford et al., Blood 2008; Hughes
et al., Blood 2006; Muller et al.,
Haematologica 2007; Muller et al.,
Leukemia 2009).
- Once my lab has become standardized to the IS, how do I maintain a valid IS
conversion factor?
Local laboratories reporting on the IS must establish their own CF to factor in
potential differences in pre-analytical and analytical steps and re-validate
their CF at minimum every 2 years or each time the procedure is changed.
(Muller et al., Leukemia 2009). Currently, and until reference standards
become broadly available, this re-validation must be performed through a sample
exchange between the local laboratory and an IS Reference Laboratory.
- Is there an IS reference material available?
Yes and no. The first World Health Organization International Genetic Reference
Panel for quantitation of BCR-ABL1 mRNA was recently established.
(White el al., Blood 2010). The WHO panel consists of 4 vials containing
freeze-dried K562 cells diluted in a background of HL-60 cells. Each vial has
been tested by 10 IS-standardized labs and the nominal IS percent ratio reported
on each vial is the average of the values obtained by the independent IS labs.
However, the reference material is in limited supply and cannot be used for
standardization of existing tests or commercial kits. “The principal function of
these primary reagents should be limited to the calibration of secondary
reference reagents.” (White et al.,
Blood 2010).
- Is there a secondary reference material traceable to the primary WHO reference material available?
The primary WHO reference material (White et al.,
Blood 2010). has been made available only to entities involved in the development of secondary reference
material anchored to the IS. The goal is to make these secondary reference materials broadly available
independently of the BCR-ABL1 assays used or commercialized by specific labs or companies. For
example, Asuragen, in collaboration with the international community, has developed a secondary IS
reference panel for research use. An initial prototype was evaluated in 29 labs worldwide and the final
configuration has been validated in IS Reference Labs. The ARQ IS Calibrator Panels are now available to a
broader number of labs, including labs not yet standardized to the IS
- How many labs have been validated on the IS?
With fewer than about 200 labs calibrated to the IS worldwide, standardization
has not yet been achieved on a global scale. In Europe, many countries have been
standardized through the establishment of National Reference Laboratories.
According to the 2010 CAP survey, fewer than a dozen US sites are standardized
to the IS.
- Which RT-qPCR methods are compatible with the IS?
A variety of laboratory-developed tests or commercial assays are compatible with
IS reporting as long as they are calibrated to the IS.
(Branford et al., Blood 2008). Different real-time technologies, instrument
platforms or assay designs can be used. In general these assays are run in
singleplex, i.e., b2a2 (e13a2), b3a2 (e14a2) and the EC’s are detected in
independent qPCR’s. Most often, b2a2 and b3a2 are co-detected in a single
reaction. Assays can be further multiplexed to co-detect BCR-ABL1 and the EC
which may improve the operational efficiency of laboratories and reduce the
number of reactions required per individual result. Co-detection of the EC also
ensures that the BCR-ABL1 and EC copy numbers are not generated by independent
reactions with different efficiencies and can be combined in a single ratio
value, independently of the number of replicates performed. For example,
Asuragen has developed a multiplex research assay that detects b2a2, b3a2, e1a2,
ABL and an exogenous process control in a single reaction while maintaining the
performance required for quantitative measurement of BCR-ABL1 on the IS.
(Brown et al., Blood Cancer Journal 2011). When running replicates, the
percent ratios are calculated as the ratio of the average copy numbers for
singleplex assays or as the average of the ratios for multiplex assays.
- Can e1a2 be reported on the IS?
No. The IRIS trial and IS standardization efforts exclusively focused on the
BCR-ABL1 fusion transcripts resulting from the major breakpoint (b2a2 and b3a2).
Therefore, there is actually no IS for e1a2 and no MMR or equivalent. Fewer than
1% of CML cases are e1a2 positive
(Verma et al., Blood 2009), which may explain the lack of data to currently
support e1a2 standardization. Additional clinical studies are required to
validate the correlation between e1a2 expression levels and long-term outcome
and define appropriate e1a2 response criteria. Standardization of e1a2
monitoring through the use of a single multiplexed assay with the same
efficiency for major (b2a2, b3a2) and minor (e1a2) BCR-ABL1 fusion transcripts
would likely be the first step toward addressing these clinical needs.
(Brown et al., Blood Cancer Journal 2011).
- Can monitoring of response be done with FISH? One can score 5,000 or
10,000 cells which will be even superior to PCR. All type of break point can be detected (MRD).
The analytical sensitivity of current FISH techniques is about 1% (1 in 100 cells). FISH can be used to
monitor CHR (complete hematologic response or 10%) and CCyR (complete cytogenetic response or 1%). Even
if one could score 10,000 cells (0.01%), monitoring of patients below MMR requires more sensitive molecular
methods. Both the NCCN and ELN guidelines recommend the use of RT-qPCR. An interesting concept that would
combine both approaches is to measure BCR-ABL11 by RT-qPCR and then normalize the result to the number of white
blood cells instead of using the copy number of an endogenous control (e.g. BCR-ABL11 copy number per million
of WBC). This can be achieved by using a process control RNA that is co-extracted with the WBC RNA and co-detected
by the RT-qPCR assay. A multiplex assay co-detecting BCR-ABL1, ABL1 and such a process control has been reported
in the literature (Brown et al. BCJ 2011). Studies are ongoing to establish the feasibility and validity of this
approach.
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