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

Ruiz, María Sol; Medina, Marcela; Tapia, Ivana Jaqueline; Mordoh, José; Cross, Nicholas C. P.; Larripa, Irene; Bianchini, Michele

doi:10.1038/leu.2016.197

Cited by 14 publications

(16 citation statements)

References 11 publications

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“…It is very important for clinicians to appropriately assess treatment response of leukemia patients by using % BCR‐ABL1 values reported on IS . For the 24 IS laboratories, the CV% of the converted result was lower and the %Lab within 2‐, 3‐, and 5‐fold were higher compared to those of the unconverted results, indicating that CFs can harmonize p210 BCR‐ABL1 transcript quantification between laboratories, consistent with previous findings . The improved comparability of p210 BCR‐ABL1 transcript values between laboratories is mainly attributed to effective work toward the standardization of p210 BCR‐ABL1 transcript quantification, such as a defined standardized baseline, the proposal of IS, establishment of CFs, and development of primary and secondary reference reagents .…”

Section: Discussionsupporting

confidence: 81%

External quality assessment of p210 BCR‐ABL1 transcript quantification by RT‐qPCR: Findings and recommendations

Zhang

et al. 2018

Int J Lab Hematology

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Section: Discussionsupporting

confidence: 81%

External quality assessment of p210 BCR‐ABL1 transcript quantification by RT‐qPCR: Findings and recommendations

Zhang

et al. 2018

Int J Lab Hematology

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“…To evaluate the minor BCR/ABL1 primer sets, we used total RNAs derived from minor and major BCR/ABL1 -negative HL60 human leukemia cell lines. [ 16 ] The HL60 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cultured cells (6×10 7 cells) were homogenized using a Tissue Ruptor (QIAGEN, Venlo, the Netherlands), and total RNA was isolated from homogenized cells using RNeasy Midi Kit (QIAGEN).…”

Section: Methodsmentioning

confidence: 99%

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

Tsuchiya

Tabe

et al. 2018

PLoS ONE

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The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called “the Eprobe leukemia assay,” for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

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“…Total RNA from PB samples was extracted using Trizol (Invitrogen, Thermo Fisher Scientific) after lysis of red blood cells. Results were harmonized to the International Scale by means of a conversion factor obtained by standardization with secondary cellular calibrators [ 28 ]. Some patients were monitored in other standardized centers, and so BCR-ABL1/ABL1 ratios were obtained directly from their data.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia

et al. 2018

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Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloid leukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI) treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a “functional leukemic burden” (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this method could be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal.

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

Cited by 14 publications

References 11 publications

External quality assessment of p210 BCR‐ABL1 transcript quantification by RT‐qPCR: Findings and recommendations

External quality assessment of p210 BCR‐ABL1 transcript quantification by RT‐qPCR: Findings and recommendations

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia

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