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BCR-ABL1 RT-qPCR for Monitoring the Molecular Response to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia

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REVIEW

BCR-ABL1

RT-qPCR for Monitoring the Molecular Response

to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia

Richard D. Press,* Suzanne Kamel-Reid,yand Daphne Ang*

From the Department of Pathology and Knight Cancer Institute,* Oregon Health & Science University, Portland, Oregon; and the Department of Pathology,y The University Health Network, Toronto, Ontario, Canada

Accepted for publication April 8, 2013.

Address correspondence to Richard D. Press, M.D., Ph.D., Department of Pathology, L113, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201. E-mail:pressr@ ohsu.edu.

The pathognomonic genetic alteration in chronic myeloid leukemia is the formation of the

BCR-ABL1 fusion gene, which produces a constitutively active tyrosine kinase that drives leukemic

transformation. Targeted tyrosine kinase inhibitor treatment with imatinib, nilotinib, dasatinib, bosutinib, and ponatinib is the cornerstone of modern therapy for this hematologic malignancy.

Real-time quantitative RT-PCR (RT-qPCR, also RQ-PCR) of BCR-ABL1 RNA is a necessary laboratory

technique for monitoring the efficacy of tyrosine kinase inhibitor therapy and quantitatively

assessing minimal residual disease. The molecular response measured by BCR-ABL1RT-qPCR assists

in identifying suboptimal responses and can help inform the decision to switch to alternative therapies that may be more efficacious (or to pursue more stringent monitoring). Furthermore, the tyrosine kinase inhibitoremediated molecular response provides valuable risk stratification and

prognostic information on long-term outcomes. Despite these attributes, informed, universal, practical utilization of this well-established monitoring test will require heightened efforts by the molecular diagnostics laboratory community to adopt the standardized reporting units of the International Scale. Without widespread adoption of the International Scale, the consensus major molecular response and early molecular response treatment thresholds will not be definable, and optimal clinical outcomes for patients with chronic myeloid leukemia may not be achieved. (J Mol Diagn 2013, 15: 565e576; http://dx.doi.org/10.1016/j.jmoldx.2013.04.007)

Virtually all patients with chronic myeloid leukemia (CML), a hematologic cancer characterized by the overproduction of immature and mature myeloid cells in the peripheral blood, spleen, and bone marrow, carry the Philadelphia chromosome (Ph), a reciprocal translocation between the Abelson gene (ABL1) on chromosome 9 and the breakpoint cluster region gene (BCR) on chromosome 22.1,2 The resulting fusion gene,BCR-ABL1, produces a constitutively active chimeric tyrosine kinase that is required to initiate, propagate, and maintain the leukemic phenotype in patients with CML. Tyrosine kinase inhibitor (TKI) therapy targets the BCR-ABL1 kinase and over the past decade has become the recommended first-line treatment approach for patients newly diagnosed in the initial chronic phase (CP) of CML. The success of TKI therapy in CML has furthermore become the scientific paradigm for molecularly targeted therapy of other cancers in the 21st century.3

Since the advent of BCR-ABL1 TKIs, patients with CML have had significantly improved prognosis, as reflected by longer median overall survival (OS) and lower rates of disease progression, compared with previous therapeutic regimens.4e6Imatinib was therst TKI approved by the US Food and Drug Administration for CML, based on the phase 3 International Randomized Study of Interferon and STI571 (IRIS) clinical trial. Long-term follow-up of CML-CP patients treated with first-line imatinib showed a progres-sion-free survival (PFS) rate of 93% at 5 years7 and an

Supported by Novartis Pharmaceuticals Corporation for medical editorial assistance.

Disclosures: R.D.P. has received honoraria from and served on advisory boards for Novartis and Ariad, has received grant support from Cepheid, and has received honoraria and grant support from and serves on an advisory board for Asuragen. S.K.-R. has received honoraria and grant support from Novartis and Bristol-Myers Squibb.

Copyrightª2013 American Society for Investigative Pathology

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estimated OS rate of 85% at 8 years (93% when only CML-related deaths were considered).8

Although most patients treated with imatinib have durable responses, resistance to imatinib does occur in some patients, most commonly through development of mutations in the BCR-ABL1 kinase domain that abrogate imatinib-induced inhibition.9e12 Dasatinib and nilotinib, more potent TKIs than imatinib, were initially approved by the Food and Drug Administration for the treatment of CML after imatinib failure or intolerance. More recently, both dasatinib and nilotinib have demonstrated, in separate phase 3 trials, superior response rates and higher rates of PFS as first-line therapy, compared with imatinib.13e17In addition, rates of major molecular response (MMR) were significantly higher with dasatinib or nilotinib versus imatinib over each of 3 years of follow-up.13,14,16,17Two other TKIs, bosutinib and ponatinib, were recently approved for second- and third-line treatment of CML.18,19

Targeted therapy with TKIs dramatically reduces the leukemic cell burden in CML.20e25 The low levels of minimal residual disease (MRD) typically achieved during TKI therapy demand a sensitive monitoring assay for reliable detection and quantitation.26Real-time quantitative RT-PCR (RT-qPCR, also RQ-PCR) has proven to be an effective and clinically validated laboratory method to quantifyBCR-ABL1

transcript levels and assess molecular responses. Further-more, given the predictive value of molecular response for long-term clinical outcomes and as a direct biomarker for drug efficacy and response, RT-qPCR has become an integral component of CML disease management.22e25

As BCR-ABL1 RT-qPCRebased molecular monitoring evolves, a number of factors have been identified as potential sources of confusion, thus limiting this assay’s practical clinical utility. In the quest to improve the labo-ratory utility of CML molecular monitoring, the following questions need to be addressed:

1. What are the clinically relevant BCR-ABL1 threshold levels for predicting TKI treatment responses and long-term PFS?

2. What are the clinically relevantBCR-ABL1threshold levels for predicting suboptimal response, loss of response, and evolving drug resistance?

3. What laboratory-specific factors limit the precision, accuracy, and sensitivity of the BCR-ABL1 RT-qPCR assay?

4. How can theBCR-ABL1RT-qPCR assay be standardized to the International Scale (IS) of measurement by which clinically relevant threshold levels have already been defined?

This review discusses the current use of molecular monitoring in the management of patients with CML receiving TKIs, its practical utility for measuring treatment response (and nonresponse), and the remaining challenges for improving molecular monitoring. The lessons learned from the extensive research on molecular monitoring of

BCR-ABL1 will benefit, not only patients with CML, but also patients with other cancers, for which a variety of molecularly targeted therapies and companion diagnostics are now becoming available.

What Are the Clinically Relevant

BCR-ABL1

Threshold Levels for Predicting TKI Treatment

Responses and Long-Term PFS?

Monitoring Response to Therapy

The clinical utility of laboratory methods for monitoring TKI therapeutic efficacy, including hematologic, cytogenetic, and PCR-based techniques, depends largely on their limits of detection. Complete hematologic response is based on normalization of peripheral blood counts [National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology (NCCN Guidelines) Chronic Myelogenous Leukemia Version 3.2013. http://www.nccn. org/professionals/physician_gls/pdf/cml.pdf, last accessed February 24, 2013; user registration required].27Cytogenetic response is based on the percentage of Ph-positive (Phþ) metaphase cells observed in a bone marrow sample, typically by Giemsa staining of metaphase chromosome spreads. Complete cytogenetic response (CCyR), a clinically impor-tant prognostic response threshold in patients with CML receiving TKIs, is defined as the absence of detectable Phþ chromosomes (NCCN Guidelines).28,29Because a minimum of 20 metaphase cells is recommended for cytogenetic eval-uation (NCCN Guidelines), the limit of detection of this technique is relatively low (1:20, or 5%). Another limitation of cytogenetic testing is the need for an invasive bone marrow biopsy to obtain culturable metaphase cells. Cytogenetic testing does have, however, the capability to detect other chromosomal abnormalities besides the Ph chromosome, and this may have prognostic relevance. Ancillary interphase cell fluorescencein situhybridization (FISH), with DNA probes forBCRandABL1,is also commonly used to monitor CML response to treatment. Because 100 to 500 interphase cells are typically examined with FISH, this approach is more sensitive (1:500 to 1:100, or 0.2 to 1%) than metaphase cytogenetics. Depending on the probes used, however, it can have a high false-positive rate. FISH lacks practical utility for MRD monitoring in the majority of TKI-treated CML patients, and therefore is not recommended by the NCCN for routine monitoring of TKI treatment response (NCCN Guidelines).

Instead, guidelines from the NCCN, the European Leu-kemiaNet (ELN),27 and the National Institutes of Health21 recommend serial BCR-ABL1 RT-qPCR assays at regular 3- to 6-month intervals for routine MRD monitoring of CML patients receiving TKI therapy. Molecular monitoring involves extraction of RNA from a bone marrow or peripheral blood specimen and subsequent RT-qPCR to measure transcript levels ofBCR-ABL1relative to those of a reference gene. Because the analyte of RT-qPCR, RNA, is labile and degradation prone, many pre-analytical variables

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can affect sample quality and quantitative results, including sample source (peripheral blood versus bone marrow), sample storage (temperature, type of tube), sample transport (duration of travel, temperature), and sample stabilization (lysis buffer, storage buffer). The reference gene in RT-qPCR serves as a control for overall RNA quality (with respect to degrada-tion) and, assuming equivalent reference gene expression in all hematopoietic cells, for the number of input cells per PCR reaction. For example, in our laboratory, when the level of reference gene RNA falls two SD below the mean, RT-qPCR for theBCR-ABL1and reference gene are repeated, and if this repeat analysis again shows low reference gene RNA levels, the sample is reported asinadequate (new sample requested). Compared with cytogenetic testing, PCR-based molecular monitoring offers exquisite analytical sensitivity, 100 to 1000 times greater than FISH or bone marrow cytogenetic analysis. It is applicable to bone marrow and peripheral blood samples, and with a short turnaround time, provides quantitative results that are associated with validated clinical response thresh-olds.30Disadvantages of RT-qPCR include its lack of meth-odological and reporting standardization, its need for specialized laboratories and equipment, and the variability of analytical and reporting systems.31 Table 1 summarizes current NCCN testing recommendations for monitoring TKI treatment response in patients with CML.

Key Levels of Molecular Response

Response to TKI therapy is assessed by the change in BCR-ABL1 transcript levels from a standardized pretreatment baseline level. The standardized baseline, defined as 100% on

the IS, was established in the IRIS study of imatinib treatment in which the three participating laboratories independently determined the median BCR-ABL1 RT-qPCR value of the same set of 30 CML-CP samples collected before imatinib treatment initiation.32Thus, 100% BCR-ABL1(IS) became the standardized baseline against which future BCR-ABL1

RNA levels were compared, and any treatment-associated reduction inBCR-ABL1would represent an absolute reduc-tion that is independent of individual patient-specific

BCR-ABL1 baseline values. MMR, a 3-log reduction in

BCR-ABL1transcript levels from this standardized baseline, is defined as 0.1% on the IS (NCCN Guidelines), and represents a clinically relevant level of molecular response, based on the observation that patients who achieved MMR had a minimal risk of disease progression and improved long-term PFS compared with patients who did not.32

Achievement of MMR has been incorporated into ELN practice guidelines to define an optimal level of response at 12 to 18 months (Table 2),27,33 and failure to achieve this molecular response milestone is considered a suboptimal response that calls for careful reevaluation of the current therapeutic regimen.27,33 Analysis of IRIS data at 7 years demonstrated that patients who had achieved MMR by 12 or 18 months had significantly superior event-free survival and OS, and more durable CCyR, compared with those who had not.24Another study showed that patients on imatinib with

2-log reduction inBCR-ABL1levels at the time of CCyR had longer PFS than patients not achieving that level of molecular response.25 In the German CML-Study IV of patients on imatinib, MMR at 12 months predicted signifi -cantly better PFS and OS at 3 years.23Further analysis of this

Table 1 Recommended Testing Parameters for Patients on TKI Therapy per NCCN Guidelines

Test Recommended test specifications Recommended test frequency

Bone marrow cytogenetics

Analyze20 metaphase cells At diagnosis

At 3 months, if RT-qPCR (IS) is not available At 12 months, if neither CCyR nor MMR is achieved At 18 months, if no MMR and no CCyR at 12 months

1-log increase inBCR-ABL1level without MMR

FISH Use peripheral blood At diagnosis, if collection of bone marrow is not feasible

Use dual probes forBCRandABL1genes Not recommended for monitoring response to treatment

RT-qPCR Use an RT-qPCR (IS) assay with sensitivity

of4.5 log below the standardized baseline

At diagnosis

Use peripheral blood or bone marrow Every 3 months for responding patients

After CCyR is achieved, every 3 months for 3 years, then every 3 to 6 months thereafter

If1-log increase inBCR-ABL1level with MMR, repeat in 1 to 3 months

BCR-ABL1KD mutational analysis

None provided If inadequate* response tofirst-line therapy or any sign

of loss of response, defined as hematologic or

cytogenetic relapse, or1-log increase inBCR-ABL1level and loss of MMR

If disease progression to accelerated or blast phase CML

*Level of response that warrants further patient evaluation and consideration of a change in therapy.

CCyR, complete cytogenetic response; CML, chronic myeloid leukemia; FISH,fluorescencein situhybridization; IS, international scale; KD, kinase domain;

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study also showed better outcomes for patients with lower

BCR-ABL1transcript levels at 3 months after TKI initiation; the 5-year OS and PFS rates were significantly longer for patients with 10% BCR-ABL1(IS) at 3 months than for patients with>10%BCR-ABL1(IS).34Attainment of a 1-log BCR-ABL1 transcript reduction, or 10% BCR-ABL1

(IS), at 3 months after TKI initiation is now defined as an early molecular response. The practical prognostic value of early molecular response has been independently confirmed in a British CML cohort, where patients who failed to attain this level of response had significantly shorter OS and PFS at 8 years than patients who did attain this early molecular response landmark.35 On the basis of these data, the most recent NCCN CML guidelines specifically recommend a change in treatment for patients who fail to reach 10% BCR-ABL1(IS) after 3 months of TKI therapy (Table 2).

Molecular response to nilotinib or dasatinib has also been shown to predict long-term outcomes. For CML-CP patients receivingfirst-line dasatinib, an early molecular response was predictive of improved long-term outcomes at 2 years.35 Similarly, CML-CP patients receiving first-line nilotinib who achieved early molecular response had higher rates of molecular response and improved PFS and OS at 2 and 3 years than patients who did not achieve early molecular response.36 In the second-line setting, patients receiving nilotinib after imatinib failure who achieved 10% BCR-ABL1(IS) at 3 months had higher 24-month event-free survival rates than patients with>10%BCR-ABL1(IS) at 3 months.37

Complete molecular response (CMR) has been defined as a level ofBCR-ABL1RNA that is undetectable by RT-qPCR. Because the absence of detectableBCR-ABL1may be due to pre-analytical factors, such as sample degradation, or to tech-nical factors, such as the variability in the limits of detection of heterogeneous laboratory-developed assays, the definition of CMR is very much sample- and laboratory-dependent. To

address these issues, it has been proposed that a finding of undetectable BCR-ABL1 be qualified by the log limit of detection of the assay as defined by the transcript level of the reference gene [eg, CMR4 Z undetectable BCR-ABL1 in a sample in whichBCR-ABL1RNA would have been detect-able, had it been present at a level above 0.01% IS (4 log below baseline)].38 In the Oregon Health & Science University (OHSU) laboratory, the cutoff sample quality threshold for defining CMR is a 4.7-log reduction from baseline [0.002%

BCR-ABL1 (IS)], as defined by the reference gene level. Approximately 10% of samples submitted to the OHSU laboratory have reference gene transcript levels below this 4.7-log threshold such that even though BCR-ABL1 may be undetectable in these samples, they are not reported as having achieved CMR. By comparison, NCCN Guidelines define

CMR as undetectable BCR-ABL1 RNA with a minimal

analytical assay sensitivity of 4.5 log below the standardized IS baseline.

Given that the achievement of10%BCR-ABL1(IS) at 3

months and 0.1% BCR-ABL1 (IS) (ie, MMR) at 18

months predict good prognosis, would achieving undetect-able BCR-ABL1 predict even better clinical outcomes? In support of this hypothesis, achieving CMR has been shown to be associated with more durable remissions and signifi -cantly longer relapse-free survival than achieving MMR without CMR.39 Other studies have shown that the frequency of CMR increases with longer treatment times, and that the achievement of MMR at 12 months predicts subsequent CMR.40 In the Stop Imatinib (STIM) study, a prospective study evaluating the feasibility of stopping imatinib treatment in patients with durable CMR, 12 months after discontinuation of imatinib, 41% of patients with

12 months of follow-up maintained CMR.41Thus, 59% of patients regained PCR-detectableBCR-ABL1RNA after ima-tinib discontinuation. The long-term prognostic significance of Table 2 Expected Levels of Response to First-line TKI Therapy (per NCCN and ELN27,33Guidelines)

Time on therapy

NCCN Guidelines (v3.2013) ELN Guidelines (2009, updated 2013)*

Adequate response Inadequate response Optimal response

Suboptimal response (“warning”) Failure (therapy change indicted) 3 Months 10%BCR-ABL1 (IS) orPCyR >10%BCR-ABL1(IS) or<PCyR CHR andPCyR;10% BCR-ABL1(IS) Ph+ 36e95%;>10% BCR-ABL1(IS) <CHR; No CyR

6 Months NS NS CCyR;<1%BCR-ABL1(IS) Phþ1e35%; 1e10%

BCReABL1(IS)

<PCyR;>10% BCR-ABL1(IS)

12 Months CCyR PCyR or cytogenetic

relapse

MMR 0.1e1%BCR-ABL1(IS) <CCyR;>1%

BCR-ABL1(IS)

18 Months CCyR PCyR or cytogenetic

relapse

Anytime NS NS Stable or improving MMR Loss of CHR, MMR, or

CCyR; KD mutations; clonal progression

*After this article was accepted, an updated version of the consensus European LeukemiaNet (ELN) recommendations for management of chronic myeloid

leukemia was published.33This table reflects those changes. Also seeNote Added in Proof.

CCyR, complete cytogenetic response (0% Phþ); ELN, European LeukemiaNet; IS, international scale; mCyR, minor cytogenetic response; MMR, major

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undetectableBCR-ABL1is actively being researched to better understand its implications.

What Are the Clinically Relevant

BCR-ABL1

Threshold Levels for Predicting Suboptimal

Response, Loss of Response, and Evolving Drug

Resistance?

If achieving MMR signals a good long-term outcome, does loss of MMR suggest impending resistance or relapse?42,43 In general, risingBCR-ABL1levels signal the need for more frequent patient follow-up, to either detect early resistance or to exclude poor adherence to TKI therapy.44 Available data suggest that a rising level of BCR-ABL1RNA during TKI therapy can indeed predict increased risk of subsequent disease progression, even if MMR has been achieved.39 Although most patients with a TKI-induced CCyR and a subsequent rise in BCR-ABL1 RNA levels remain in CCyR, there is a persistent increased risk of both relapse and a newly developed BCR-ABL1 kinase domain (KD) muta-tion,39,43,45 which, by itself, is a proven risk factor for disease progression.4 ELN guidelines consider a rise in

BCR-ABL1levels at any time during treatment to constitute a warning of a possible suboptimal response (Table 2).27,33 At the very least, rising BCR-ABL1levels should warrant a repeat RT-qPCR within the next 1 to 3 months (NCCN Guidelines), sooner than the recommended routine testing interval, the goal being early detection of true disease progression, and possible intervention before full-fledged cytogenetic or hematologic relapse.

How high must BCR-ABL1 RNA levels rise to be of clinical concern? Cutoff values to define a clinically significant rise inBCR-ABL1RNA levels vary by laboratory and therefore have been difficult to standardize. Suggested transcript rise thresholds that should prompt clinical action have included a 0.5-log increase,39a 1-log increase (NCCN Guidelines), and a twofold (0.3-log) increase.47 A subse-quent study using a receiver-operating characteristic curve analysis found that a 2.6-fold (0.41-log) increase in BCR-ABL1 levels was the optimal cutoff for predicting the presence of a concomitant KD mutation.43 The negative predictive value of this 2.6-fold cutoff was 97%, suggesting that only 3% of patients with transcript rises<2.6-fold will have a mutation that is missed. This study also showed that had the NCCN Guidelineerecommended 10-fold (1-log) transcript rise cutoff been used as a trigger for KD mutation analysis, the diagnostic sensitivity would have been poor (26%), with many confirmed mutations missed. Recom-mendations from the ELN suggest direct mutation testing when primary treatment with imatinib fails, when the increase inBCR-ABL1transcripts leads to loss of MMR, or whenever there is a suboptimal response (eg, lack of MMR after 18 months of imatinib).12 Updated NCCN Guidelines recommend mutation testing when the initial response is inadequate [no partial cytogenetic response or >10%

BCR-ABL1(IS) at 3 months, no CCyR at 12 or 18 months], when there is any sign of loss of response (hematologic or cytogenetic relapse, or 1-log increase inBCR-ABL1and loss of MMR), or when CML progresses to advanced stages of disease.

BCR-ABL1

KD Mutations Directly Inform

Therapeutic Choices

The emergence of BCR-ABL1 KD mutations has been

shown to be associated with an increased likelihood of subsequent disease progression in patients with TKI-treated

CML.43,46Although the switch to an alternative TKI,

trig-gered by the finding of a KD mutation, has never been

shown to directly improve long-term outcomes in

a prospective trial, ELN and NCCN guidelines nevertheless specifically recommend a switch to certain TKI agents when particular mutations are detected.12Due to the diversity of mutations, full BCR-ABL1KD mutation screening is done in most laboratories by a direct Sanger DNA sequencing technique that has a detection limit of approximately 20% mutant allele.11,48A significant majority ofBCR-ABL1KD mutations cluster to one of four hot spots: the ATP-binding P-loop (amino acids 248 to 256); the imatinib-binding region (amino acids 315 to 317); the catalytic domain (amino acids 350 to 363); and the activation (A)-loop (amino acids 381 to 402).11 Differential sensitivity to ima-tinib, dasaima-tinib, niloima-tinib, bosuima-tinib, and ponatinib has been demonstrated by these diverse mutant BCR-ABL1 kinases in in vitro studies.49e52 Because there is often, but not always, a good correlation between mutation-specificin vi-tro resistance andin vivo clinical responses for some, but not all, KD mutations and TKIs, the identification of the specific mutation can help to inform the optimal manage-ment strategy.11,12,49 In particular, the presence of the common T315I mutation suggests that ponatinib, and no other TKI, may be effective.19In addition, NCCN and ELN guidelines suggest a switch to nilotinib (not dasatinib) for patients with theV299L,T315A, orF317L/V/I/Cmutations; and a switch to dasatinib (not nilotinib) for patients with the

Y253H,E255K/V, orF359V/C/Imutations.

Aside from point mutations, the BCR-ABL1 KD also commonly develops insertion/deletion mutations, including a 35-bp intronic insertion at the exon 8 to 9 junction, an

L248Vmutation with deletion of 81 bp of exon 4, an exon 7 deletion, and several others.53e55Although the clinical and drug resistance significance of most of these insertion-deletion mutations is still unclear, the very common 35-bp intronic insertion after exon 8 does not appear to mediate TKI resistance,in vitroorin vivo.56TheBCR-ABL1KD also carries some common single nucleotide polymorphisms that appear to be wholly benign, including three nonsynonymous (K247R, F311V, Y320C), and three synonymous (T240T,

T315T,E499E) variants, each of which has no known effect on TKI binding or drug resistance.57

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What Laboratory-Speci

c Factors Limit the

Precision, Accuracy, and Sensitivity of the

BCR-ABL1

RT-qPCR Assay?

The dearth of high-quality, affordable, standardized reagents remains a substantial barrier to wider adoption of IS-standardized BCR-ABL1 RT-qPCR assays. Assay-related heterogeneity affects all aspects of the multistep RT-qPCR procedure (Table 3), which contributes to the considerable variability inBCR-ABL1quantitative values reported by US clinical diagnostic laboratories [College of American Pathologists (CAP) Surveys 2012 and Anatomic Pathology Education Programs. MRD participant summary report, hereinafter referred to as CAP Survey].58 Several studies have indicated a need for improvement inBCR-ABL1 RT-qPCR assay reproducibility (precision) between laborato-ries and even within laboratolaborato-ries.58e62 For example, in a study of 38 laboratories that used blinded shared samples,

BCR-ABL1RT-qPCR results varied by 1.6 to 3 log for the

same sample.58 A more comprehensive and less biased source of real-world data on BCR-ABL1 RT-qPCR analyt-ical assay performance is the CAP MRD proficiency survey whereby blinded CML RNA samples are sent biannually to participating clinical molecular diagnostic laboratories as part of a proficiency testing program required to maintain laboratory accreditation (CAP Survey). Over each of the past 3 years (2009, 2010, 2011), one CAP MRD survey per year has included the same 1 in 10,000 (4-log) dilution of CML cell-line K562 RNA, along with a comparative sample containing undiluted K562 RNA. In these surveys, CAP has required that laboratories report a relative log reduction of the BCR-ABL1 RNA ratio in the diluted (post-treatment) sample divided by the undiluted (baseline) sample. Instead of the expected 4-log difference between samples, and Table 3 Sources of Heterogeneity among US Laboratories in

BCR-ABL1RT-qPCR Assessment

Source of heterogeneity inBCR-ABL1RT-qPCR measurement

Equipment and reagents

No predominant real-time PCR instrument (brand and model) used in most laboratories

Multiple sources of PCR primers and probes: commercially designed versus laboratory designed

Different primers for reverse transcription: random hexamers versus gene-specific primers

Multiple manufacturers/distributors of reverse transcriptase and Taqpolymerases

Methodology

No predominant RNA extraction method used across all laboratories

Variability in RT-qPCR protocols: commercially available kit versus laboratory-developed assay

No predominant laboratory-developed assay used across all laboratories

No predominant reference gene used across all laboratories: ABL1,ACTB,B2M,BCR,G6PD,GAPDH,GUSB,TBP

Multiple types of material used to generate standard curve (or no standard curve generated): dilutions derived from plasmid DNA, RNA, cDNA, cell lines

Multiple sources of patient material used in RT-qPCR replicates: RNA, cDNA, cells, cell extracts/lysates

Reported results

No predominant method for reporting RT-qPCR results used across all laboratories;BCR-ABL1level reported as:

Copies per unit of RNA

Ratio, relative to control gene, laboratory median or mean of diagnostic samples, patient’s previous result, diagnostic patient sample, diluted or undiluted K562 cells, or other Log reduction from pre-treatment baseline

Percentage on the IS

Table is compiled from Zhang et al58and the CAP MRD Survey.

BCR-ABL Log-Reduction from Baseline

Number of CAP Laboratories 45 40 35 30 25 20 15 10 5 0 -2 -1 0 1 2 3 4 5 6 7 N: 300 labs Mean: 3.3 SD: 0.99 Median: 3.4 25-75%: 2.7 – 3.8 ABL (n = 187) Non-ABL (n = 111) Reference Gene p < 0.0001

BCR-ABL Log-Reduction from Baseline

-2 -1 0 1 2 3 4 5 6 7

Figure 1 Distribution ofBCR-ABL1 RT-qPCR log-reduction values in

laboratories participating in the CAP MRD proficiency testing survey. Once in

each of the years 2009, 2010, and 2011, approximately 100 laboratories

received two blinded samples for BCR-ABL1RT-qPCR analysis: a baseline

sample containing undiluted K562 cell-line RNA and a post-treatment sample containing K562 RNA (dilution 1:10,000 in non-CML cell RNA).

Each laboratory measured theBCR-ABL1RNA ratio by its own validated

method, and reported back to CAP the relative log reduction of the

post-treatment sample compared with the baseline sample.A: Histogram of the

combined log-reduction data from the cumulative 3-year testing period (nZ

300).B: Box-whisker plot of the log-reduction values split by the reference

genes used, eitherABL1(nZ187) or any other non-ABL1reference gene (n

Z111). Information regarding reference gene use was not available for two

laboratories. Thearrowon thexaxis indicates the expected target

log-reduction value (4.0). Thevertical linein the middle of each box is the

median value of the distribution. Thenotcharound the median represents

the 95% CI for the median. Thevertical linesat the end of each box are the

25% and 75% values of the distribution. Thevertical barsoutside of each

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despite efforts to improve assay standardization and preci-sion by eliminating method-dependent variability from the log-reduction result calculation, the distribution of reported log-reduction values (nZ300) showed considerable assay imprecision and inaccuracy (Figure 1A). The overall mean log reduction of 300 reported results from three combined CAP surveys (2009, 2010, 2011) was 3.3 log (median, 3.4 log), 0.7 log removed from the expected 4-log value. There was substantial interlaboratory variability (0.99 log SD) in this cumulative 3-year distribution that was not considerably different from that observed in the individual surveys [0.8 log SD in 2009 (nZ87),1.0 log in 2010 (nZ108), and

0.9 log in 2011 (n Z 105)]. This high degree of inter-laboratory imprecisiondeven without accounting for vari-ability stemming from RNA extraction and other method biasesdclearly precludes the practical ability to meaning-fully interpret serial BCR-ABL1 RNA levels when those measurements are not all performed in the same laboratory, and reaffirms the critical need for improved assay stan-dardization. Furthermore, these data suggest that many clinical laboratories are inaccurate in their quantification of

BCR-ABL1 RNA, with 32% of laboratories reporting log-reduction values >1 log (10-fold) removed from the ex-pected 4-log value.

A significant and much-debated source of variability in the BCR-ABL1 RT-qPCR assay is the choice of reference gene used to calculate the relative transcript ratio.58,63 In a direct exploration of this issue, Wang et al63 examined nine reference genes and deemedGUSBas the most suitable for BCR-ABL1 RT-qPCR. In another study, Zhang et al58 found BCR to be a more suitable reference gene than

GUSB. Nonetheless, of the seven reference genes used by approximately 100 CAP-surveyed laboratories, the two

most commonly used are ABL1 and G6PD, together

accounting for over 80% of laboratories (CAP Survey).

AlthoughABL1is by far the most popular reference gene, it is actually a rather poor choice given that the PCR primers typically used for quantitating ABL1 also cross-react and amplify BCR-ABL1.58,63,64 Thus, the measured level of

ABL1actually reflects levels ofBCR-ABL1plusABL1, and fluctuates in samples with varying leukemia burdens. These sample-specific fluctuations in reference gene RNA levels are completely contradictory to the primary defining char-acteristic of an ideal RT-qPCR reference gene. Thus, the resulting BCR-ABL1/ABL1 RNA ratio is nonlinear and falsely low, particularly when the leukemia burden is high.63 When the ABL1 reference gene is used for monitoring during TKI treatment, the log-reduction from baseline calculation will be skewed artificially low, and the resulting IS value therefore will be falsely high. Consistent with this prediction, the blinded CAP survey data showed that for the 187 laboratories usingABL1as the reference gene, the log-reduction values (mean, 2.9 log 0.07 SE) were signifi -cantly lower (by 0.9 log; P < 0.0001) than for the 111

0.5 N = 29 shared samples Before Conversion: 13/29 within 2-fold 18/29 within 3-fold 26/29 within 5-fold Bias = 0.35 logs (P < 0.001)

Conversion Factor = 2.22 (anti-log bias)

IS units (OHSU) = (BCR-ABL ratio)* 2.22 MMR level at OHSU (0.1% IS) = 0.045%

Bias = 0.35 X = Y 0.0 -0.5 Adelaide IS (log) -1.0 -1.5 -2.0 -2.5 -2.0 -1.5 -1.0 BCR-ABL Ratio (log)

-0.5 0.0 0.5 -2.5 0.5 N = 29 shared samples After Conversion: 15/29 within 2-fold 27/29 within 3-fold 29/29 within 5-fold

Bias = 0.025 logs (1.06-fold)(P = 0.69) No significant bias Validated CF = 2.22 X = Y 0.0 -0.5 Adelaide IS (log) -1.0 -1.5 -2.0 -2.5 -2.0 -1.5 -1.0 BCR-ABL IS (log) -0.5 0.0 0.5 -2.5

Figure 2 IS CF determination at the Oregon Health & Science University (OHSU; Portland, Oregon). Twenty-nine samples were shared between OHSU and

the IS-validated Hughes & Branford laboratory (Adelaide, Australia). Each laboratory independently measuredBCR-ABL1RNA in these samples, and a log-log

linear regression analysis (bold line) was used to determine the OHSU laboratory’s CF to the IS (anti-log of bias).A: Before conversion to the IS.B: After

conversion to the IS, using the laboratory-specic CF of 2.22. The XZY line (thin and gray) indicates a perfect correlation between the methods.

Percentage of CAP Labs

30 25 20 15 10 5 0 2009A Year: CAP labs, N:

2009B 2010A 2010B 2011A 2011B 2012A 100 108 122 129 136 143 156

2012B 154

Figure 3 Percentage of CAP laboratories usingBCR-ABL1RT-qPCR IS

reporting. Surveys are repeated early (A) and late (B) each calendar year.

Data are self-reported by the laboratories subscribing to the CAP MRD survey.

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laboratories using any other non-ABL1 reference gene (mean, 3.8 log0.08 SE) (Figure 1B). In other words, the average laboratory using the ABL1 reference gene falsely overquantitated BCR-ABL1 RNA by >1 log (>10-fold) relative to the expected 4-log result. Although this ABL1

reference geneedependent assay bias is perhaps exacer-bated in this CAP survey that utilizes a cell line (K562) with a knownBCR-ABL1gene amplification, an analogous (but perhaps lower magnitude) overquantitation phenomenon would also be expected to occur with any other sample source.

How Can the

BCR-ABL1

RT-qPCR Assay Be

Standardized to the IS of Measurement, by

Which Clinically Relevant Threshold Levels

Have Already Been De

ned?

Inadequate clinical laboratory adoption of a standardized reporting scale forBCR-ABL1RT-qPCR remains a signifi -cant barrier to the real-world implementation of the consensus molecular monitoring clinical practice guidelines recently put forth in both the US and Europe (NCCN Guidelines).27 International efforts to better standardize

BCR-ABL1RT-qPCR reporting have been ongoing for many years, and have recently resulted in at least two notable achievements: the creation of an IS of quantitative measurement forBCR-ABL1RT-qPCR21and the availability of standardized reference material calibrated to the IS and validated by the World Health Organization (WHO).65

The original samples used in the IRIS study to establish the anchor values (ie, the pretreatment median standardized baseline and MMR) for the IS are no longer available, but quantitative traceability to those original values has been provided by the Hughes & Branford laboratory in Adelaide, Australia, which participated in the IRIS study and has maintained strict assay quality control standards for the past decade.26,31 Because the BCR-ABL1 IS anchor values represent absolute, not relative, values and are not dependent on the pretreatment baseline value of any particular patient,

laboratories around the world, using heterogeneous methods for RT-qPCR (including different reference genes), can adopt the IS without fundamental technical changes to local stan-dard operating procedures.31 Two specific methods are available for harmonizing a laboratory’s local BCR-ABL1

RT-qPCR assay to the IS: sample sharing with a laboratory already harmonized to the IS to establish a mathematical conversion factor (CF),31 and local assay calibration with reference material that is calibrated to the IS.65

The sample-exchange process for conversion to the IS involves an exchange of samples with a validated reference laboratory whose BCR-ABL1 RT-qPCR assay is already aligned to the IS. The referenceBCR-ABL1RT-qPCR data from this reference laboratory are compared with those generated in afield laboratory, and a CF is mathematically derived from simple linear regression and bias analyses.31 This CF is a constant fixed number by which the RT-qPCR ratio generated in the field laboratory is multiplied to convert to the IS. In a study seeking to align the BCR-ABL1RT-qPCR values of 38field laboratories from around the world to that of the IS-calibrated Adelaide laboratory, calculated CFs ranged from 0.18 to 13.5 in an initial round of sample exchanges. In our laboratory, the CF to the IS was 2.22 (Figure 2). With a second round of sample exchanges, the majority offield laboratories were able to validate their CFs, with performance characteristics such that <10% of patients who achieved MMR would be misclassified.31

Although this sample exchange approach successfully attained its goal of minimizing interlaboratory BCR-ABL1

reporting heterogeneity, several issues remain to be addressed: i) For laboratories that exchange RNA (and not raw cell lysates), how should different RNA preparation methods be controlled for? ii) How stable are the CFs? iii) How often should the sample exchange procedure be repeated? and iv) What degree of change to the technical protocol (eg, different PCR reagent manufacturing lots) should trigger a recalculation of the CF? Furthermore, the sample exchange process is challenging, time consuming, and expensive. The practical difficulty of this process is reflected in serial data from the CAP MRD BCR-ABL1

Figure 4 The pathway for use of certied reference standards forBCR-ABL1molecular monitoring, illustrating traceability back to primary standards

certied by the WHO. Based on White et al64. The actual assigned IS values for the WHOBCR-ABL1reference material are slightly different from the 10%, 1%,

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proficiency testing survey (CAP Survey), which shows that, despite the proven analytical and clinical advantages of the IS, only a minority of clinical laboratories have successfully switched to this reporting method (Figure 3). Although adoption of the IS among clinical laboratories has increased over the past 3 years, only 45 of 154 (29%) laboratories surveyed in late 2012 used IS-based reporting (up from 4% in 2009). This trend of increased adoption of IS-based reporting, likely due to the recent commercial availability of BCR-ABL1 RT-qPCR assay reagents that are directly calibrated to the IS, will likely continue. In the interim, however, for CML patients being serially monitored in those 71% of laboratories not using the IS, it is unclear how the efficacy of TKI therapy is being clinically assessed, given the consensus IS-defined BCR-ABL1RNA level thresholds for early response and MMR.

The availability of internationally validated, traceable, widely available universal reference materials would greatly facilitate the conversion of BCR-ABL1 testing laboratories to the IS. Toward this goal, a WHO International Genetic Reference Panel was recently established and validated for quantitation of BCR-ABL1 RNA with any of three commonly used control genes (ABL1,BCR, andGUSB).65 These extensively tested reference materials consist of a lyophilized admixture of K562 and HL60 cell lines at four different dilution levels, with BCR-ABL1 RNA levels that are close to clinical thresholds (eg, MMR) and directly tied to the IS.65 Unfortunately, only a limited supply of this primary reference material was created, and it is envisioned that reference laboratories, commercial reagent vendors, and other organizations will use this primary material to create validated large-scale secondary reference materials to be used as routine calibrators in individual testing laboratories (Figure 4).65 Several commercial reagent vendors now market such IS-standardized reagents or calibrators. In addition, two of these vendors have each recently begun clinical trials with the aim of providing Food and Drug Administrationeapproved assay kits that will automatically incorporate IS-based reporting units for BCR-ABL1.

Conclusions

It is now widely accepted that molecular monitoring by serial

BCR-ABL1RT-qPCR provides essential information on TKI treatment response in patients with CML. In particular, the achievement of MMR at 18 months of TKI therapy is recog-nized as a consensus indicator of successful treatment response and subsequent good prognosis. More recently, the NCCN guidelines have included the achievement of early molecular response, defined as10%BCR-ABL1(IS) at 3 months post-TKI initiation, as an important response milestone that directly informs disease management decisions at this early time point. Additional data suggest that even lower levels of MRD, namely CMR (undetectable BCR-ABL1using an RT-qPCR assay with sensitivity of4,4.5, or5 log), may afford

even better prognosis. The failure to achieve optimal thera-peutic landmarks, as defined by BCR-ABL1 (IS) values, conveys an increased risk of poor outcome, and may indicate the need to switch the TKI agent. Furthermore, increasing levels ofBCR-ABL1RNA indicate a rising leukemia burden and confer an increased risk of disease progression and the appearance of drug-resistant mutations inBCR-ABL1. Regular monitoring (every 3 to 6 months) with accurate, reproducible, and standardizedBCR-ABL1RT-qPCR assays is thus required throughout the course of TKI therapy. Imprecision and inac-curacy in measuringBCR-ABL1transcripts can have serious adverse clinical consequences, and treatable problems of early relapse or resistance may go unnoticed.

Efforts to facilitate accurate and precise measurements of

BCR-ABL1RNA were enhanced recently by the validation of WHO reference standards. It is hoped that the recent availability of validated secondary reference materials based on the primary WHO reference standards will encourage more laboratories to adopt the standardized IS for reporting of BCR-ABL1 assay results. This should facilitate the accurate interlaboratory comparison of data, and the consistent identification of patients who have achieved clinically relevant therapeutic milestones such as early molecular response or MMR (or even CMR). Improved reliability of molecular monitoring will enhance the confi -dence in these measurements to the benefit of clinicians, laboratories, pathologists, patients, regulators, and payers.

Note Added in Proof

While this manuscript was in preparation for print, the European LeukemiaNet (ELN) published updated recom-mendations for management of chronic myeloid leukemia.33 These new ELN recommendations vary from the 2009 ELN recommendations primarily by the addition of specific time-dependent molecular monitoring thresholds for defining “Optimal,” “Warning” (previously called “Suboptimal”), and“Failure”categories of TKI responses. In particular, the 2013 ELN recommendations update the information in Table 2of this manuscript to include:

An“Optimal”response is defined by a 3-month post-TKI BCR-ABL qPCR value below 10% IS, a 6-month post-TKI BCR-ABL RT-qPCR value below 1% IS, and a 12-month post-TKI BCR-ABL RT-qPCR value below 0.1% IS (ie, MMR).

A “Failed” response (meaning a change of therapy is recommended) is defined by a 6-month post-TKI BCR-ABL qPCR value above 10% IS and a 12 month post-TKI BCR-ABL qPCR value above 1% IS.

Acknowledgments

We thank Drs. Anna Lau and Patricia Segarini (Percolation Communications LLC) for their medical editorial assistance,

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Dr. Lawrence Jennings (Lurie Children’s Hospital, Chicago, IL) for his insightful preview of the manuscript, and Mariana Ovnic for the illustrations inFigure 4.

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References

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