Odk-1201, One-Step RT-qPCR Major BCR-ABL/ABL mRNA Kit for the International Scale, with High Sensitivity to Detect Deeper MR
Authors: Matsumura Itaru , Nakamae Hirohisa , Yoshida Chikashi , Fletcher Linda , Branford Susan , Koga Daisuke , Sogabe Takayuki , Kanakura Yuzuru , Naoe Tomoki 
Published: October 2019

Abstract
Background:
Molecular monitoring by the measurement of major BCR-ABL mRNA levels is important for the evaluation of the therapeutic efficacy of TKI in CML patients. However, there is no commercial kit easily available in each laboratory or hospital, which can estimate deeper responses below MR4.5that is 4.5-log reduction by the international scale (IS) 0.0032%.
Methods:
An ODK-1201 kit (Otsuka Pharmaceutical Co. Japan) is based on a HawkZ05 Fast One-Step RT-PCR Kit (Roche, Indianapolis, IN, USA). In this kit, reverse transcription and quantitative PCR (RT-qPCR) are performed in one tube, of which products are measured by ABI 7500 fast Dx (Life Technologies Japan, Japan). To isolate enough amount of RNA for the evaluation of molecular responses below MR4.5in CML patients, 7 ml of peripheral blood is required for this kit.
To get the conversion factor (CF) for the IS, the panel of WHO International Standard was obtained from NIBSC, which comprises four ampoules each containing freeze-dried BCR-ABL-positive and -negative cells at various ratios from 0.01% to 10%. RNAs were isolated from each ampoule with a QIAamp RNA Blood Mini Kit (QIAGEN) and were subjected to the measurement of major BCR-ABL and ABL mRNAs with an ODK-1201 kit. Since %BCR-ABL/ABL in the WHO standard panel was already defined, CF was calculated by comparing the values of %BCR-ABL/ABL obtained by ODK-1201 with those of WHO standard panel.
To validate the results obtained by ODK-1201, we collected 267 peripheral blood samples from CML patients as a multicenter study, and 120 samples in these were measured by ODK-1201 and in the international reference laboratory (Adelaide Lab), respectively. In addition, we conducted another multicenter study to compare ODK-1201 with an Ipsogen BCR-ABL1 Mbcr IS-MMR DX Kit (QIAGEN) and a One-Step qRT-PCR BCR-ABL Kit (Molecular MD), in which we measured the samples from 201 CML patients, 25 patients with non-CML hematologic malignancies and 25 healthy volunteers by these methods. All of the samples were obtained after the written informed consent was given.
Results:
The CF of ODK-1201 determined by the WHO standard panel was 1.12. After conversion to the IS by this CF, %BCR-ABL/ABLIS determined by ODK-1201 ranged from 0.0007% to 144.6867% in CML patients. In contrast, all of the values were below 0.0007% in the samples from patients with non-CML hematologic malignancies and healthy volunteers.
The overall correlation between ODK-1201 and Adelaide Lab system was very good (94% within 3-fold bias) and the evaluation of MMR was concordant in 84% of CML samples. As for 46 CML samples with %BCR-ABL/ABL IS between 0.0032% and 0.1%, IS values by ODK-1201 were similar to the ones by Adelaide Lab system (with MR4.5; 0.0032%) with 89% within 3-fold and high correlation coefficient (r=0.89). Also, the correlation between ODK-1201 and Ipsogen was r = 0.97 (n = 224) and that between ODK-1201 and Molecular MD kit was r = 0.97 (n = 221), indicating that ODK-1201 was as accurate as Ipsogen and Molecular MD kits. Together, these results indicate that ODK-1201 was capable of measuring at least MR4.5 with the lowest value detected as 0.0007% (calculated sensitivity of less than MR5.0) by the IS.
Conclusions:
We developed a major BCR-ABLmRNA kit, ODK-1201 with CF for the IS and confirmed its high sensitivity (0.0007%) and accuracy. The procedure is very simple and takes only 2.5 h, and it needs a small amount of blood because RT-qPCR is performed in a single tube. It will enable us to evaluate deeper molecular responses in any laboratories.
Conflict of interest :received research fundinand and honoraria from Otsuka
I.M. received honoraria from Otsuka during the conduct of the study. H.N. received research funding from Otsuka during the conduct of the study and received research funding and honoraria from otsuka outside the submitted work. C.Y. received research funding from Otsuka during the conduct of the study and honoraria from Novartis and Bristol Myers Squibb. S.B. received research funding and honoraria from Novartis, Bristol Myers Squibb, Otsuka and Ariad. L.F. has no conflict of interest. K.D. and T.S. are employee of Otsuka. Y.K. received honoraria from Otsuka during the conduct of the study. T.N. received grants and personal fees from Otsuka during the conduct of the study, and grants and personal fees from Otsuka outside the submitted work.

Disclosures
Matsumura: Otsuka: Consultancy, Honoraria. Nakamae:Otsuka : Research Funding. Yoshida:Otsuka: Research Funding. Branford:Otsuka: Research Funding. Koga:Otsuka: Employment. Sogabe:Otsuka: Employment. Kanakura:Otsuka: Consultancy. Naoe:Otsuka: Consultancy.