Residual disease in chronic myeloid leukemia (CML) patients undergoing therapy with tyrosine kinase inhibitors (TKIs) is measured by assessing the quantity of transcripts of the BCR-ABL1 fusion gene in peripheral white blood cells. This analysis is based on reverse-transcription quantitative PCR (RT–qPCR) technology; however, the wide array of methods used worldwide has led to large variation in quantitative BCR-ABL1 measurements, which hamper inter-laboratory comparative studiesFil: Ruiz, María Sol. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Medina, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Tapia, I.. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Mordoh, Jose. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Cross, N. C. P.. Universidad de Southampton Uk; Reino UnidoFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Bianchini, Michele. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentin

Bianchini, Michele

Cross, N. C. P.

Larripa, Irene Beatriz

Medina, M.

Mordoh, Jose

Ruiz, María Sol

Tapia, I.

Leukemia

English

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

1  Title Standardization of molecular monitoring for chronic myeloid leukemia in Latin America using locally produced secondary cellular calibrators Running title Latin America RT-qPCR standardization platform Authors María Sol Ruiz1, Marina Medina2, Ivana Tapia1, José Mordoh1, Nicholas C.P. Cross3,4, Irene Larripa2, Michele Bianchini1 1Centro de Investigaciones Oncológicas–Fundación Cáncer (CIO-FUCA), Instituto Alexander Fleming, Buenos Aires, Argentina. 2Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina, Buenos Aires, Argentina. 3Faculty of Medicine, University of Southampton, Southampton, UK. 4National Genetics Reference Laboratory (Wessex), Salisbury, UK  Corresponding author: Dr. Michele Bianchini Instituto Alexander Fleming CIO-FUCA Zabala 2836 - 1426 Buenos Aires, Argentina. Telephone:+54-11-3221-8900 e-mail:mbianchini@conicet.gov.ar  Conflict of interest: This study was supported financially by a Novartis grant to IL and MB. MB and NCPC have received speaker fees from Novartis; all other authors declare no competing financial interests.  ©    2016 Macmillan Publishers Limited. All rights reserved.2  Residual disease in chronic myeloid leukemia (CML) patients undergoing therapy with tyrosine kinase inhibitors (TKIs) is measured by assessing the quantity of transcripts of the BCR-ABL1 fusion gene in peripheral white blood cells1. This analysis is based on reverse-transcription quantitative PCR (RT-qPCR) technology; however, the wide array of methods used worldwide has led to large variation in quantitative BCR-ABL1 measurements, which hamper inter-laboratory comparative studies2,3. It is now recognized that monitoring BCR-ABL1/control gene ratios on the International Scale (IS) is vital for the management of patients with CML4. Efforts to harmonize procedures to measure BCR-ABL1 fusion transcripts have included important investments in sample exchange programs to derive laboratory-specific conversion factors (CF); these efforts showed improvements in inter-laboratory concordance rates, but the process is laborious and limited due to the lack of a common set of reference samples that can be shared on a global scale. This requirement was recently addressed in part by the formulation and validation of the first World Health Organization (WHO) International Genetic Reference Panel for quantitation of BCR-ABL1 by RT-qPCR5. The WHO primary standards consist of a 4-level panel of e14a2-positive lyophilized cell line dilutions. Each level has an assigned IS-value, which was obtained by repeated testing of each sample level in expert IS-standardized laboratories. Unfortunately, the stock of WHO primary standards is limited, and their accessibility has been restricted to manufacturers of testing kits or secondary reference standards. In this study, we aimed to develop and validate secondary reference materials calibrated to the IS through the WHO primary standards in order to facilitate standardization of molecular monitoring in Latin America.  The study design comprised five principal steps as illustrated in Supplementary Figure 1. The study was conducted by a single reference laboratory (rLAB, Buenos Aires, Argentina), which initially obtained a CF to the IS by sample exchange with the reference laboratory in Adelaide in 2010. To ensure consistent performance of rLAB analytical system (MolecularMD, OR, USA), we included 2 quality control (QC) RNA-samples with a high and low BCR-ABL1 level in each run ©    2016 Macmillan Publishers Limited. All rights reserved.3  (Supplementary Table 1 and Supplementary Material and Method, Section 1). To further validate our method prior to calibration of secondary reference materials (see below), we derived a CF by using WHO primary standards (NIBSC code 09/138) from the United Kingdom National Institute for Biological Standards and Control (Potters Bar, UK). The antilog of the estimated mean bias (-0.152) was designated as the conversion factor (CF=0.7) for the rLAB method (Supplementary Table 2), very close (i.e. well within 2 fold) to the value of 0.45 obtained by sample exchange 3 years previously. Previous local exploratory investigation indicated us that RT-qPCR methodologies harmonization was necessary since the tests were inadequately comparable, considering that the three acceptance criteria proposed by Muller et al.6 were not satisfied (Supplementary Tables 3 and 4). To this aim we established and validated five batches of cellular calibrators produced by serial dilution of the Ph-positive cell line K562 in the Ph-negative cell line HL-60. Formulations were planned to target IS% ratios close to each of the established TKI clinical response criteria7,8,9, that is, between 10%, 1%, 0.1% (MR3.0), 0.01% (MR4.0). An additional dilution (0.001%, MR5.0) was included in order to assess the limit of detection of the methods and was not considered for the estimation of the CF (Figure 1). Cell mixes were stabilized by lyophilisation (Supplementary Figure 2A); assignment of IS%-values to each batch and level of these secondary standards was achieved by repeated testing of randomly picked ampoules on 4 non-consecutive days (Figure 1 and Supplementary Material and Method, Section 2). Stability studies of the freeze-dried cells, showed no significant changes in the BCR-ABL1/ABL1 ratio over time at different temperatures up to six months (Supplementary Figure 2B). The calibrated secondary reference standards were distributed to 18 testing laboratories from 7 countries in Latin America. Each laboratory followed the calibrators’ instructions for use, which recommended 4 independent runs on different days for the two panels (Supplementary Materials and Methods, Section 3). A total of 1,312 RT-qPCR positive results were generated; mean raw percentage ratios generated in all laboratories were consistent ©    2016 Macmillan Publishers Limited. All rights reserved.4  with a 10-fold serial dilution and were linear for the first 4 levels (Supplementary Figure 3); prior to CF calculation, in order to assess if bias was uniform across the BCR-ABL1 expression range, Bland-Altman analysis10 was performed for each assay (Supplementary Material and Method, Section 4). All the assays showed a uniform bias, indicating that a valid CF could be calculated for these methods (Supplementary Table 5). All raw %ratios measured within the linear range of each local RT-qPCR method were compared against a single set of reference values, the nominal IS%-ratios. Laboratory-specific CFs were calculated by Bland–Altman method10 (Supplementary Table 5). The relative mean bias ranged from -0.45-fold to +0.25-fold in distinct laboratories (Figure 2A); after correction of the individual raw percent ratios with the laboratory specific CF, the residual mean bias was null (Figure 2B). In addition, raw BCR-ABL1/ABL1 %ratios generated in each laboratory were used to assess the corresponding level-specific coefficient of variation (CV): between 8% and 58% (12 out of 18 laboratories with an average CV less than 30%). As expected, BCR-ABL1 was inconsistently detected below the linear quantitative detection range of the RT-qPCR method in the lowest positive samples (fifth calibrator); for the 10 laboratories that tested the fifth calibrator, the overall BCR-ABL1 detection rate ranged from 12.5% to 100%. In total, eight out of ten laboratories could reproducibly detect BCR-ABL1 in level-5 samples (Supplementary Table 5). Interestingly, 5 laboratories had previous standardization, thus we could compare the new CF to the laboratory’s current CF; when no methodology modifications were introduced since the time of the last calibration we obtained highly concordant CFs (Supplementary Table 6). For appropriate clinical decisions, we need to judge agreement between the methods after IS conversion; to this aim, we interchanged 41 whole blood samples (divided into two parts) from CML patients with Lab#09. The concordance in MRIS between the reference method and the external laboratory was 88% after conversion (36 out of 41 samples were in the same MR category) (Figure 2C). This result underlines the importance of conversion to the IS, given that, after harmonization we were able to halve the number of discordant data (from 9 to 5 cases). ©    2016 Macmillan Publishers Limited. All rights reserved.5  In the present study, we show that secondary reference biological calibrators anchored to the WHO primary standards can decrease inter-laboratory variability. Our results, together with those recently reported by Cross et al.11, substantiate the objective initially set during the establishment of the WHO primary standards, that is, to facilitate worldwide diffusion of the IS. For the first time in Latin America, this study provides a platform on which to assess the performance of distinct clinical BCR-ABL1 tests and confirm the utility of secondary reference materials to further improve IS accuracy and inter-laboratory precision. This effort will continue in the future by providing secondary reference material to the centres involved in this project and potential new participants; moreover, due to its higher precision and absolute quantification capability, we are evaluating to include digital PCR as the calibration method for the future. Acknowledgements: We thank Novartis Oncology for its support to this initiative. The Latin America BCR-ABL1 Standardization (LABS) study group consists of: Marina Gutierrez (Stamboulian); Isabel Giere (Fundaleu), Guillermo Bramuglia (Argenomics); Marta Alonso (Hospital Posadas); Evangelina Agrielo (LEB); Mariana Asinari (Hospital Privado); Gabriel Via (Biogen); Margarita Bragos (Hospital Centenario); Maria Monaco, (LabTuc); Analia Serravalle (CIBIC); Maria Altuna (Clinica Raña); Gonzalo Manrique (Sociedad Española), Javier Gonzalez (MeyerLab), Carlos Santamaria (Hospital Nacional de Niños), Yaribeth Olmedo (ION), Luisa Rosales (INVEGEN) and Olga Zea (GeneticaLab). Conflict of interest: This study was supported financially by a Novartis grant to IL and MB. MB and NCPC have received speaker fees from Novartis; all other authors declare no competing financial interests. Supplementary Information is available at Leukemia’s website   ©    2016 Macmillan Publishers Limited. All rights reserved.6  Bibliography 1- Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J et al. European Leukemia Net. Chronic myeloid leukemia: an update of concepts and management recommendations of European Leukemia Net. J Clin Oncol 2009; 27(35): 6041-51. 2- Branford S, Cross NC, Hochhaus A, Radich J, Saglio G, Kaeda J et al. Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia. Leukemia 2006; 20(11): 1925-30. 3- Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF et al. European Leukemia Net recommendations for the management of chronic myeloid leukemia: 2013. Blood 2013; 122(6): 872-84. 4- Branford S, Fletcher L, Cross NC, Müller MC, Hochhaus A, Kim DW et al. 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(8): 3330-8. 5- White HE, Matejtschuk P, Rigsby P, Gabert J, Lin F, Lynn Wang Y et al. Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA. Blood 2010; 116(22): e111-7. 6- Müller MC, Cross NC, Erben P, Schenk T, Hanfstein B, Ernst T et al. Harmonization of molecular monitoring of CML therapy in Europe. Leukemia 2009; 23(11):1957-63. 7- Cross NC, White HE, Müller MC, Saglio G, Hochhaus A. Standardized definitions of molecular response in chronic myeloid leukemia. Leukemia 2012; 26(10): 2172-5.  8- Cross NC, White HE, Colomer D, Ehrencrona H, Foroni L, Gottardi E et al. Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia. Leukemia 2015; 29(5):999-1003.  ©    2016 Macmillan Publishers Limited. All rights reserved.7  9- Morley AA. Reporting results for deep molecular responses in chronic myeloid leukemia. Leukemia 2016; e-pub ahead of print 17 May 2016; doi: 10.1038/leu.2016.94 10- Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1(8476):307-10. 11- Cross NC, White HE, Ernst T, Welden L, Dietz C, Saglio G, et al. Development and evaluation of a secondary reference panel for BCR-ABL1 quantitation on the International Scale. Leukemia 2016; e-pub ahead of print 25 April 2016; doi: 10.1038/leu.2016.90 Figure legends Figure 1: Study design for the nominal IS% value assignment and BCR-ABL1 testing by participating laboratories. Figure 2: (A) Relative difference between nominal and measured IS% ratios for all calibrator levels combined (black circle). (B) Residual difference after CF-conversion. Error bars show 95% LOA interval. (C) Correlation graph. Comparison of IS%-ratios from 41 whole blood samples, interchanged between the rLAB and local Lab#09. Grey-coloured squares represent the concordance area for the assignment of the molecular response. Black points; concordant results. Red points; discordant results. Black line; equality line. ©    2016 Macmillan Publishers Limited. All rights reserved.Figure 1©    2016 Macmillan Publishers Limited. All rights reserved.ABCFigure 2©    2016 Macmillan Publishers Limited. All rights reserved.

Standardization of molecular monitoring for chronic myeloid leukemia in Latin America using locally produced secondary cellular calibrators

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Standardization of molecular monitoring for chronic myeloid leukemia in Latin America using locally produced secondary cellular calibrators

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