Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA
2019; Elsevier BV; Volume: 21; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2019.03.002
ISSN1943-7811
AutoresJustin T. Brown, Ion Beldorth, Walairat Laosinchai-Wolf, Marie E. Fahey, Keri L. Jefferson, Adam K. Ruskin, Jacquelyn J. Roth, Cai Li, Christopher D. Watt, Richard D. Press, Fei Yang, John Hedges, Bernard F. Andruss,
Tópico(s)Acute Lymphoblastic Leukemia research
ResumoThis study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Food and Drug Administration–cleared assay designed to monitor breakpoint cluster region–Abelson tyrosine-protein kinase 1 (BCR-ABL1) fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex real-time quantitative RT-PCR assay amplifies both e13a2 and e14a2 Major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided in international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the World Health Organization BCR-ABL1 Primary Reference Materials, without the necessity of determining and maintaining conversion factors. For each batch run, the integrated interpretive software evaluates run and specimen quality control metrics (including a sufficient amount of ABL1 control transcripts to ensure a minimal limit of detection) and calculates both molecular response (MR) and %IS values for each specimen. The test has a limit of detection of MR4.7 (0.002%IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7). The performance of this BCR-ABL1 monitoring test meets all of the clinical guideline recommendations for sensitivity and IS reporting for the management of chronic myeloid leukemia patients. This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Food and Drug Administration–cleared assay designed to monitor breakpoint cluster region–Abelson tyrosine-protein kinase 1 (BCR-ABL1) fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex real-time quantitative RT-PCR assay amplifies both e13a2 and e14a2 Major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided in international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the World Health Organization BCR-ABL1 Primary Reference Materials, without the necessity of determining and maintaining conversion factors. For each batch run, the integrated interpretive software evaluates run and specimen quality control metrics (including a sufficient amount of ABL1 control transcripts to ensure a minimal limit of detection) and calculates both molecular response (MR) and %IS values for each specimen. The test has a limit of detection of MR4.7 (0.002%IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7). The performance of this BCR-ABL1 monitoring test meets all of the clinical guideline recommendations for sensitivity and IS reporting for the management of chronic myeloid leukemia patients. There are approximately 1.8 newly diagnosed cases of chronic myeloid leukemia (CML) per 100,000 individuals per year, with the median age at diagnosis of 65 years.1Noone A.M. Howlader N. Krapcho M. Miller D. Brest A. Yu M. Ruhl J. Tatalovich Z. Mariotto A. Lewis D.R. Chen H.S. Feuer E.J. Cronin K.A. SEER Cancer Statistics Review, 1975-2015. National Cancer Institute, Bethesda, MD2018Google Scholar CML accounts for approximately 10% to 15% of all adult cases of leukemia. The genetic hallmark of all cases of CML is the reciprocal translocation between the long arms of chromosomes 9 and 22, termed t(9; 22) (q34.1; q11.2), generating a fusion gene breakpoint cluster region–Abelson tyrosine-protein kinase 1 (BCR-ABL1) on the derivative chromosome 22 (alias the Philadelphia chromosome).2Faderl S. Talpaz M. Estrov Z. O'Brien S. Kurzrock R. Kantarjian H.M. The biology of chronic myeloid leukemia.N Engl J Med. 1999; 341: 164-172Crossref PubMed Scopus (1035) Google Scholar Most of the translocations occur between the Major breakpoint cluster region of BCR and the intron upstream of exon 2 of ABL1. The Major breakpoint cluster region occurs downstream of either exon 13 or exon 14 of BCR and results in the formation of the Major fusion transcript e13a2 or e14a2, coding for a 210-kDa protein often referred to as p210. Major fusion transcripts (ie, p210) account for >95% of CML cases, and patients can exhibit both species of Major BCR-ABL1 transcripts.3Quintas-Cardama A. Cortes J. Molecular biology of bcr-abl1-positive chronic myeloid leukemia.Blood. 2009; 113: 1619-1630Crossref PubMed Scopus (493) Google Scholar In less than approximately 1% of CML (and two-thirds of Philadelphia-positive acute lymphoblastic leukemia), the translocation localizes to the minor breakpoint cluster region of BCR, resulting in an e1a2 transcript that produces a 190-kDa protein.3Quintas-Cardama A. Cortes J. Molecular biology of bcr-abl1-positive chronic myeloid leukemia.Blood. 2009; 113: 1619-1630Crossref PubMed Scopus (493) Google Scholar Furthermore, a 230-kDa protein is observed in rare CML cases from translocation e19a2 and is designated as the micro breakpoint.3Quintas-Cardama A. Cortes J. Molecular biology of bcr-abl1-positive chronic myeloid leukemia.Blood. 2009; 113: 1619-1630Crossref PubMed Scopus (493) Google Scholar Even rarer variants have been identified, including translocations at exon 3 of ABL1.4Arun A.K. Senthamizhselvi A. Mani S. Vinodhini K. Janet N.B. Lakshmi K.M. Abraham A. George B. Srivastava A. Srivastava V.M. Mathews V. Balasubramanian P. Frequency of rare BCR-ABL1 fusion transcripts in chronic myeloid leukemia patients.Int J Lab Hematol. 2017; 39: 235-242Crossref PubMed Scopus (20) Google Scholar, 5Burmeister T. Reinhardt R. A multiplex PCR for improved detection of typical and atypical BCR-ABL fusion transcripts.Leuk Res. 2008; 32: 579-585Crossref PubMed Scopus (48) Google Scholar In all cases, the resulting BCR-ABL1 fusion protein is a constitutively active kinase that can drive uncontrolled proliferation in myeloid precursor cells, causing the clinical manifestation of CML. The specific structural characteristics of the chimeric BCR-ABL1 protein allowed for the generation and subsequent Food and Drug Administration (FDA) approval of several tyrosine kinase inhibitors (TKIs) designed to inhibit the intrinsic kinase activity of the ABL1 moiety of the hybrid protein.6Jabbour E.J. Cortes J.E. Kantarjian H.M. Tyrosine kinase inhibition: a therapeutic target for the management of chronic-phase chronic myeloid leukemia.Expert Rev Anticancer Ther. 2013; 13: 1433-1452Crossref PubMed Scopus (20) Google Scholar The ability of these TKIs to reduce the number of leukemic cells in CML patients to levels far below previous chemotherapy- or interferon-based treatments has driven demand for a more sensitive, validated molecular monitoring method to track the dynamics of the disease. The pivotal International Randomized Study of Interferon and STI571 (IRIS) trial established quantitative RT-PCR (RT-qPCR) as the laboratory method of choice for monitoring the reduction of BCR-ABL1 transcripts in TKI-treated CML patients.7Hughes T.P. Kaeda J. Branford S. Rudzki Z. Hochhaus A. Hensley M.L. Gathmann I. Bolton A.E. van Hoomissen I.C. Goldman J.M. Radich J.P. International Randomised Study of Interferon versus STISG: frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.N Engl J Med. 2003; 349: 1423-1432Crossref PubMed Scopus (1056) Google Scholar On the basis of this and many other studies, RT-qPCR is considered the gold standard method of monitoring BCR-ABL1 transcripts, as recommended by internationally recognized guidelines for the management of CML (National Comprehensive Cancer Network, https://www.nccn.org/professionals/physician_gls/PDF/cml.pdf, last accessed February 20, 2018).8Baccarani M. Deininger M.W. Rosti G. Hochhaus A. Soverini S. Apperley J.F. Cervantes F. Clark R.E. Cortes J.E. Guilhot F. Hjorth-Hansen H. Hughes T.P. Kantarjian H.M. Kim D.W. Larson R.A. Lipton J.H. Mahon F.X. Martinelli G. Mayer J. Muller M.C. Niederwieser D. Pane F. Radich J.P. Rousselot P. Saglio G. Saussele S. Schiffer C. Silver R. Simonsson B. Steegmann J.L. Goldman J.M. Hehlmann R. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013.Blood. 2013; 122: 872-884Crossref PubMed Scopus (1416) Google Scholar Despite the clinical successes achieved with FDA-approved targeted therapies in CML, there remains the widespread challenge of consistently and reproducibly monitoring BCR-ABL1 transcript levels because of the variation in the design and performance characteristics of research-use-only reagents and laboratory-developed tests. To align patient monitoring to established clinical milestones and to address the portability of patient-specific data across methods and laboratories, the National Institutes of Health Consensus Group recommended the standardization of BCR-ABL1 monitoring using an international scale (IS).9Hughes T. Deininger M. Hochhaus A. Branford S. Radich J. Kaeda J. Baccarani M. Cortes J. Cross N.C. Druker B.J. Gabert J. Grimwade D. Hehlmann R. Kamel-Reid S. Lipton J.H. Longtine J. Martinelli G. Saglio G. Soverini S. Stock W. Goldman J.M. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (973) Google Scholar The IS is a numeric scale anchored to the standardized baseline established in the IRIS trial (100%IS) with a 3-log reduction from baseline defined as a major molecular response (MMR; MR3; 0.1%IS).7Hughes T.P. Kaeda J. Branford S. Rudzki Z. Hochhaus A. Hensley M.L. Gathmann I. Bolton A.E. van Hoomissen I.C. Goldman J.M. Radich J.P. International Randomised Study of Interferon versus STISG: frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.N Engl J Med. 2003; 349: 1423-1432Crossref PubMed Scopus (1056) Google Scholar Laboratory harmonization was established, validated, and repeatedly revalidated through sample exchange with an IS-aligned laboratory, generating a laboratory-specific conversion factor that, when applied to results from that laboratory's RT-qPCR test, aligned patient results to the IS.10Branford S. Fletcher L. Cross N.C. Muller M.C. Hochhaus A. Kim D.W. Radich J.P. Saglio G. Pane F. Kamel-Reid S. Wang Y.L. Press R.D. Lynch K. Rudzki Z. Goldman J.M. Hughes T. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials.Blood. 2008; 112: 3330-3338Crossref PubMed Scopus (298) Google Scholar Subsequently, the National Comprehensive Cancer Network (https://www.nccn.org/professionals/physician_gls/PDF/cml.pdf) and the European LeukemiaNET8Baccarani M. Deininger M.W. Rosti G. Hochhaus A. Soverini S. Apperley J.F. Cervantes F. Clark R.E. Cortes J.E. Guilhot F. Hjorth-Hansen H. Hughes T.P. Kantarjian H.M. Kim D.W. Larson R.A. Lipton J.H. Mahon F.X. Martinelli G. Mayer J. Muller M.C. Niederwieser D. Pane F. Radich J.P. Rousselot P. Saglio G. Saussele S. Schiffer C. Silver R. Simonsson B. Steegmann J.L. Goldman J.M. Hehlmann R. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013.Blood. 2013; 122: 872-884Crossref PubMed Scopus (1416) Google Scholar incorporated reporting BCR-ABL1 transcripts directly on the IS into the clinical guideline recommendations for managing CML patients. To allow harmonization between methods, the World Health Organization then established the first World Health Organization International Genetics Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR (National Institute for Biological Standards and Control code 09/138) in limited quantities.11White H.E. Matejtschuk P. Rigsby P. Gabert J. Lin F. Lynn Wang Y. Branford S. Muller M.C. Beaufils N. Beillard E. Colomer D. Dvorakova D. Ehrencrona H. Goh H.G. El Housni H. Jones D. Kairisto V. Kamel-Reid S. Kim D.W. Langabeer S. Ma E.S. Press R.D. Romeo G. Wang L. Zoi K. Hughes T. Saglio G. Hochhaus A. Goldman J.M. Metcalfe P. Cross N.C. Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.Blood. 2010; 116: e111-e117Crossref PubMed Scopus (118) Google Scholar This study describes the performance characteristics of the first US FDA-cleared molecular test to monitor BCR-ABL1 transcripts in CML patients. The test reports results in both the MR scale and %IS through the use of traceable armored RNA reference materials and using automated software with integrated quality control algorithms. Over 7300 data points were generated on RNA extracted from human peripheral blood specimens to validate the test's performance characteristics, including >3600 such data points across studies for limit of detection (LOD; MR4.7), limit of quantitation (LOQ; MR4.7), linearity (from MR0.3 to MR4.7), limit of blank (LOB; undetected), and both single-site and multisite precision across four independent laboratories. Furthermore, the test is sufficiently precise to rely on singleton testing for each patient specimen using RNA isolated via typical methods from specimens up to 72 hours after venipuncture. Results included in this report were generated using RNA derived from CML-positive human blood specimens, nonleukemic human blood specimens, or cell line cultures. Blood specimens were obtained with patient consent under a clinical protocol under institutional review board approvals. Blood specimens were collected in EDTA anticoagulant and isolated within 72 hours of venipuncture. Leukocytes were counted by hematological analysis using a COULTER Ac·T diff Analyzer (Beckman Coulter Inc., Brea, CA) to ensure sufficient material for each study was processed into RNA. Specimens were combined with five volumes of Erythrocyte Lysis Buffer (Qiagen, Germantown, MD), incubated at room temperature for 5 minutes, and then centrifuged at 500× g at 4°C for 5 minutes to pellet remaining cells. Cell pellets were resuspended in two volumes of Erythrocyte Lysis Buffer, incubated an additional 5 minutes at room temperature, and again centrifuged for 5 minutes at 500 × g at 4°C. These leukocyte pellets were lysed in an appropriate amount of TRIzol (Thermo Fisher Scientific, Waltham, MA) to yield the equivalent of approximately 2 × 107 cells/mL, unless otherwise noted (see RNA Isolation Method). Lysates were then frozen at ≤−70°C until further processing. Unless otherwise noted, RNA was purified from blood at various scales, according to the manufacturer-recommended, isopropanol-based precipitation protocol for TRIzol, scaled appropriately for bulk isolation. RNA quality and quantity were analyzed using a NanoDrop ND-1000 (Thermo Fisher Scientific). All reaction components were provided within the QuantideX qPCR BCR-ABL IS Kit (Asuragen, Inc., Austin, TX) and used in accordance with the Instructions for Use (US FDA clearance DEN160003), summarized herein. Each kit contains sufficient reagents for 60 reaction wells across a maximum of four uses. If a single run is performed, up to 49 specimens can be analyzed alongside the 11 calibrator and control wells. Briefly, the calibrators and controls included with the kit are based on Armored RNA Quant (ARQ) technology.12Stevenson J. Hymas W. Hillyard D. The use of Armored RNA as a multi-purpose internal control for RT-PCR.J Virol Methods. 2008; 150: 73-76Crossref PubMed Scopus (25) Google Scholar Four calibrators are composed of blends of BCR-ABL1 and ABL1 RNA targets to recapitulate the CT values observed in the kit with the World Health Organization primary reference materials. RNA materials were designed to control for the relative batch run efficiency of both the RT and PCR processes. Three controls are formulated to BCR-ABL1 content that is at a high (>1%IS, MR<2), low ( 3), or negative (ABL1 only) fusion transcript level. These materials were heat lysed and then equilibrated to room temperature. A total of 5 μL of RT Master Mix (3.5 μL per sample of RT Buffer plus 1.5 μL per sample of RT Enzyme Mix) was distributed to reaction wells on a Fast Optical 96-well plate (Applied Biosystems by Thermo Fisher Scientific), followed by 10 μL of either Kit Calibrators (prepared in duplicate), Kit Controls (in singleton), or test specimens (1000 to 5000 ng per reaction in singleton, unless otherwise required by the specific study design). RT was performed on 7500 Fast Dx Real-Time PCR instruments (Applied Biosystems by Thermo Fisher Scientific) and run in standard mode with the following program: 25°C for 10 minutes, 42°C for 45 minutes, 93°C for 10 minutes, then hold at 25°C for up to 60 minutes. The qPCR setup was initiated during this final 60-minute hold step. For qPCR, a 15-μL qPCR Master Mix containing 11 μL per sample of qPCR Buffer, 3.4 μL Test Primer/Probe Mix, and 0.6 μL qPCR Enzyme Mix was added to each intended-use well on a new 96-well reaction optical plate, followed by 10 μL of cDNA product from the RT. Thermal cycling was performed on 7500 Fast Dx Real-Time PCR instruments in standard mode with the following thermal cycling conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 63°C for 1 minute, collecting data on the Cy5 (for ABL1) and FAM (for BCR-ABL1) channels, and using the ROX channel as a passive reference dye. Other instruments that contain these fluorescence channels might be used with appropriate validation. However, differences in optical system design and thermal cycling technology may yield unanticipated performance shifts. All batch run files generated by the 7500 Fast Dx system were analyzed using 21 CFR Part 11 Module 7500 Fast Real-Time PCR System Sequence Detection software version 1.4.1 (Applied Biosystems by Thermo Fisher Scientific). The background fluorescence baseline cycles and threshold levels are determined using the following parameters: Cy5 (ABL1) using a manual threshold of 0.05 and a manual baseline from cycle 5 to 13 and FAM (BCR-ABL1) using auto threshold and auto baseline. SDS files were then directly processed (without export) through QuantideX qPCR IS Kit software version 1.1 (Asuragen, Inc.) to extract the CT of each reaction, generate the standard curve, automatically assess quality control pass/fail criteria for the batch run and specimens, and calculate the result of each test specimen. Results detailed in this study are from runs that passed all quality control criteria for the calibrators, controls, and test specimens (eg, sufficient specimen ABL1 to ensure a minimal LOD for the system). The acceptance criteria for each batch run include automated review of the controls, which are also disclosed in the test system's instructions as follows: high control, MR≤2.0 (≥1%IS); low control, MR≥3.0 (≤0.1%IS); and negative control, undetected (sufficient ABL1). For cases in which any one of these three conditions was not met, the batch run was considered invalid and no data were reported for the unknown specimens. This is discussed further in Overall Performance, in Results. The QuantideX IS software reports both MR values (MR in logs of reduction from the international baseline of 100%IS, or MR0) and %IS by linear regression to an IS-aligned, four-point, four-log standard curve of ΔCT(BCR-ABL1)-(ABL1) versus MR. Although most publications originally assigned MR values in bins (eg, MR4, MR4.5, or MR5 as a scoring system in Cross et al13Cross N.C. White H.E. Colomer D. Ehrencrona H. Foroni L. Gottardi E. Lange T. Lion T. Machova Polakova K. Dulucq S. Martinelli G. Oppliger Leibundgut E. Pallisgaard N. Barbany G. Sacha T. Talmaci R. Izzo B. Saglio G. Pane F. Muller M.C. Hochhaus A. Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia.Leukemia. 2015; 29: 999-1003Crossref PubMed Scopus (219) Google Scholar), the concept of a continuous MR scale was introduced early in the kit's development. As such, the primary output of the standard curve is MR, using lot-specific calibrator values traced to the World Health Organization primary reference set, and requires no further correction or conversion factor for alignment to the IS. Each %IS was calculated automatically by the software as the antilog of MR (ie, %IS = 102−MR). All statistical analyses were performed on the primary MR output and, therefore, all statistical results (eg, SDs) are in the same log10 scale. The %IS measurement is a historical convention well understood in the field. However, data are not normally distributed when reported on this scale, and a long tail is often observed. Where reported, analysis of %IS was performed after a log10 transformation of the data because it is normally distributed after such transformation and the assumptions of general statistical methods therefore apply. Results are converted back to the arithmetic %IS scale using Equations 1 and 2, which are required to properly calculate the mean and SD when using data transformed from normal to nonnormal distributions (ie, SD of MR to SD of %IS). The random variable X = %IS and Y = log10(X). The mean of %IS (μx) is calculated using Equation 1 from the mean and variance of the log10 (%IS) values (μy and σy2). The equations for mean (Equation 1) and variance (Equation 2) are from a report by Quan and Zhang.14Quan H. Zhang J. Estimate of standard deviation for a log-transformed variable using arithmetic means and standard deviations.Stat Med. 2003; 22: 2723-2736Crossref PubMed Scopus (51) Google Scholar Base 10 was substituted for each instance of natural base (e) in conformance with the scale of MR value.14Quan H. Zhang J. Estimate of standard deviation for a log-transformed variable using arithmetic means and standard deviations.Stat Med. 2003; 22: 2723-2736Crossref PubMed Scopus (51) Google ScholarMean.μx=eμy+σy22(1) Variance.σx2=(μx)2(eσy2−1)(2) With the exceptions of analytical specificity (exclusivity) and RNA isolation method (see below for each), all test specimens were either human CML-positive clinical specimens (whole blood stability), human CML-negative specimens (LOB), or human diagnostic-level (ie, MR<1.0) CML-positive clinical specimens diluted into human CML-negative specimens (all other studies). A single RNA panel was used for both single-site precision studies (Asuragen, Inc.) and multisite precision studies (Oregon Health and Science University, Portland OR; Hospital of the University of Pennsylvania, Philadelphia, PA; Laboratory Corporation of America, Research Triangle Park, NC; and Asuragen, Inc.). Specimens were composed of five RNA isolates from residual clinical CML-positive whole blood specimens, each serially diluted into RNA from CML-negative whole blood. The CML-negative RNA samples were used either individually or mixed to achieve five distinct backgrounds of sufficient quantity and biological variability. Therefore, the panel consisted of five dilution series—two series of e13a2, two of e14a2, and one mixed e13a2/e14a2—with five target MR values per series (MR1, MR2, MR3, MR3.5, MR4; or 10%IS, 1%IS, 0.1%IS, 0.032%IS, and 0.01%IS, respectively), for a total of 25 unique specimens. Actual values were determined from data generated across 20 total runs by three operators using three kit lots and three qPCR instruments and testing five samples in duplicate per run (single-site precision) or with two operators at each of three sites across 5 days testing 25 samples in duplicate per run to investigate site- and lot-specific variability (primary arm of multisite precision). A second arm of the multisite precision study was performed at a fourth site to investigate operator- and day-specific variability. RNA concentration for each panel member was normalized to 3000 ng/RT, the middle of the kit's required input range. In total, 200 measurements were generated for the single-site precision study (n = 20 runs × 5 samples × 2 replicates = 200) and 1200 measurements were generated for the multisite precision study (arm 1: n = 3 sites × 5 days × 25 samples × 2 replicates = 750; arm 2: n = 3 operators × 3 runs × 25 samples × 2 replicates = 450). Data were analyzed using a random effect analysis of variance using the lmer function (https://www.rdocumentation.org/packages/lme4/versions/1.1-19/topics/lmer) in R software version 3.2.2 (The R Project for Statistical Computing, https://www.r-project.org). The means and SDs were calculated using unmodified mean and SD functions in R. All samples targeted to the same MR value were grouped as replicates, giving n = 40 at each of the five levels. The SD values from individual potential sources of variation are reported in Supplemental Tables S1 and S2. The precision study's acceptance criteria were derived by a requirement of the test to distinguish specimens at the clinically relevant cut point of MR3 from those at one log lower analyte level at MR4. This supports assessment of the relapse definition of a 1-log increase in BCR-ABL1 with concomitant loss of MMR (MR3.0) (https://www.nccn.org/professionals/physician_gls/PDF/cml.pdf). For example, assuming independent error for the two samples, and SDs of 0.21 and 0.29 for samples at MR3.0 and MR4.0, respectively, the SD of the difference in measured MR values would be 0.212+0.292=0.36, implying that a 95% CI for the difference in MR between the two samples would exclude 0 (ie, MR3.0 ± 0.36 and MR4.0 ± 0.36 do not overlap, and the samples can therefore be distinguished). Although this criterion defined acceptable imprecision at this low level of analyte that approaches the anticipated LOD, greater precision is desired at higher %IS values and attainable with this technology.10Branford S. Fletcher L. Cross N.C. Muller M.C. Hochhaus A. Kim D.W. Radich J.P. Saglio G. Pane F. Kamel-Reid S. Wang Y.L. Press R.D. Lynch K. Rudzki Z. Goldman J.M. Hughes T. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials.Blood. 2008; 112: 3330-3338Crossref PubMed Scopus (298) Google Scholar Therefore, the three bins of acceptable performance were developed in Table 1, wherein more stringent criteria are set for higher analyte levels. The MR3 and MR4 levels discussed above fall within the lower two bins. The imprecision of the single MR value is compounded from two separate measurements, each with its own level of error: BCR-ABL1 and ABL1. Consistent with data performance representation standards from the US FDA (FDA Office of Regulatory Affairs, Office of Regulatory Science, https://www.scribd.com/document/329743903/ora-laboratory-manual, last accessed September 21, 2018), the ratio of these two measurements is only as precise as the least specific quantification included in its calculation. Therefore, the accumulated variability embedded in an MR value will be equivalent to or higher than that from a single measure with a comparable level of precision.Table 1Study Criteria for PrecisionLog scaleLinear scaleMR valueSD criteria%IS value∗Shown for reference; SD values of MR measurements formed the definitive acceptance criteria.% CV 0.0316≤503.5–4.25≤0.290.0316–0.0056≤75>4.25≤0.36<0.0056≤100Data expressed as %IS are not normally distributed. However, MR values are normally distributed, facilitating statistical analyses. The %IS values above are shown to bridge to historical perspectives only. The estimated % CV for the %IS values were calculated to be equivalent to the SD criteria set for the MR space.IS, international scale; MR, molecular response.∗ Shown for reference; SD values of MR measurements formed the definitive acceptance criteria. Open table in a new tab Data expressed as %IS are not normally distributed. However, MR values are normally distributed, facilitating statistical analyses. The %IS values above are shown to bridge to historical perspectives only. The estimated % CV for the %IS values were calculated to be equivalent to the SD criteria set for the MR space. IS, international scale; MR, molecular response. Specimens were composed of RNA from nonleukemic human whole blood specimens presumed to be negative or undetectable for the Major BCR-ABL1 breakpoints detected by the test. RNA isolation followed the same conditions used for patient specimens (see RNA Isolation Method). The study was designed to test the LOB of the test system, which begins with RNA extracted via a laboratory-validated method. The RT and qPCR steps for all batch runs included the standard set of calibrators and controls. Thirty specimens from unique donors, ranging from 1000 to 5000 ng/RT, were tested across multiple kit lots, operators, calendar days, and qPCR instruments, yielding 265 valid test results. Each of the nine batch runs contained a singleton of each of the 30 specimens. Any specimen that had sufficient ABL1 without detectable BCR-ABL1 (ie, no CT value within the 40 qPCR cycles performed) was considered undetected for the transcripts of the Major BCR-ABL1 translocations. Analysis followed EP17-A2 as a guide.15CLSIEvaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline-Second Edition. CLSI document. Clinical and Laboratory Standards Institute, Wayne, PA2012Google Scholar The section "Probit Approach," whereby LOB was assumed to be 0 (ie, for a qPCR assay, a sample putatively ne
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