An MMR Control RNA for Reliable Monitoring of BCR-ABL Transcripts in Treated CML Patients.
Authors: Toplin Julie , Fuller Courtney , Fletcher Linda , Wong Stephane , Maslak Peter , Cortes Jorge , Mazanet Rosemary , Branford Susan , Hughes Timothy , Galderisi Chad , Heinrich Michael , Druker Brian , Beillard Emmanuel 
Published: October 2019

Abstract
Introduction: Major Molecular Response (MMR) is defined as a three-log reduction from a standardized baseline of BCR-ABL/control gene transcript ratio in CML patients at diagnosis. MMR has prognostic significance for progression-free survival for patients on Imatinib® therapy. Day-to-day monitoring of the MMR value in clinical laboratories is challenging due to the absence of a commercially available standardized MMR control RNA. To improve the reliability of BCR-ABL quantitation, MolecularMD has evaluated the feasibility of a single MMR control RNA valid for blood samples drawn in EDTA or PAXgene™ tubes.
Material and Methods: Patient sample RNAs were interchanged between our laboratory and an International Randomized Interferon versus STI571 study (IRIS) laboratory, which had established an MMR value and international scale reporting. This exchange enabled our laboratory to establish an MMR value and reporting on an international scale using a validated conversion factor. A serial dilution of a BCR-ABL positive cell line into a human BCR-ABL negative cell line was prepared. These dilutions were tested in IRIS laboratories with established MMR value and international scale reporting and at our laboratory by QRT-PCR to determine the BCR-ABL/control gene ratio using respectively BCR and ABL control genes. We compared the BCR-ABL/ABL ratio in 104 paired PB CML patient samples drawn either in EDTA and PAXgene tubes and the BCR-ABL/BCR ratio in 32 patient samples. Stability studies were performed to evaluate the degradation of liquid and dried forms of the MMR RNA.
Results: We established a conversion factor (CF) of 0.81 with an MMR value of 0.123%. Using this CF and MMR value, we created appropriate RNA dilutions that matched the MMR value using ABL as a control gene. Repeated analyzes of this MMR control RNA confirmed the accuracy of the sample with a median value of 0.124%, very close to the MMR value defined previously (0.123%). Stability studies demonstrated that the dried RNA samples could be stored several days at 37°C and freeze-thawed up-to 10 times without significant degradation. These RNA samples once reconstituted with water could also be used several times for BCR-ABL monitoring without any significant degradation. Comparison of BCR-ABL/ABL ratio between EDTA and PAXgene tubes revealed differences unlikely to have clinical impact on disease management suggesting that the MMR RNA created would be suitable under both EDTA and PAXgene extraction methodologies.
Conclusions: We produced a stable MMR control RNA in large quantity for accurate monitoring of the MMR value. This MMR control RNA is now be tested in several laboratories to confirm the stability and reliability of this reagent. The MMR control RNA will be an important tool for standardizing MMR value in laboratories, and an integral part of a BCR-ABL QRT-PCR diagnostic kit.