Project 2: Genomic PCR

Patient-specific bcr-abl1 genomic fusion analysis of minimal residual disease of cml patients eligible for tki stopping significantly outperformed mrna detection either by qpcr or digital pcr

Katerina Machova Polakova* 1, Hana Zizkova1, Eliska Motlova1, Pavla Pecherkova1, Jan Zuna2, Lenka Hovorkova2, Olga Zimmermannova2, Tomas Jurcek3, Katerina Vlcanova1, Hana Klamova1, Nicholas C.P. Cross4, 5, Andreas Hochhaus6, Thomas Ernst6 1Institute of Hematology and Blood Transfusion, 2CLIP, Dept. of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, 3Center of Molecular Biology and Gene Therapy, Internal Hematology and Oncology Clinic, Faculty Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic, 4Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, 5Faculty of Medicine, University of Southampton, Southampton, United Kingdom, 6Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, University of Jena, Jena, Germany

Background: Data from clinical studies on stopping tyrosine kinase inhibitor (TKI) treatment of eligible patients with chronic myeloid leukemia (CML) show that a TKI cessation treatment protocol is safe under strict conditions and 40-60% of patients may sustain treatment free remission. The critical issue of the protocol is the sensitive and precise monitoring of deep molecular response (DMR)/minimal residual disease (MRD). The ELN recommended the use of standardized qPCR of Major BCR-ABL1 transcripts for MRD assessment; a limitation of qPCR is accurate detection and quantification of rare targets. Published data indicates that digital PCR may enable more sensitive and accurate assessment of DMR. Furthermore, DNA assays detecting patient specific BCR-ABL1 genomic fusions may uncover the presence of residual CML cells in cases with undetectable BCR-ABL1 transcripts.

Aims: To assess differences of MRD detection of BCR-ABL1 by 4 approaches in CML patients who responded to TKI treatment with sustained DMR.

Methods: BCR-ABL1 DNA fusions were identified in 78 CML patients. TKI was stopped in 14/78 patients within the EURO-SKI study. So far, DNA specific assays for quantification by qPCR and droplet digital PCR (ddPCR) have been optimized for 42 patients. DNA and RNA were extracted from the same number of leukocytes per sample of peripheral blood (PB) from regularly scheduled monitoring in practice or EURO-SKI study. Bone marrow (BM) was collected at the time of sustained DMR in 7 patients to follow differences in MRD detection between BM and PB. For each approach applied, mRNA qPCR (1), DNA qPCR (2), mRNA ddPCR (3) and DNA ddPCR (4), we established stringent quality requirements to exclude poor quality samples from evaluations (Table 1). So far, we analyzed 829 paired PB samples with BCR-ABL1 transcripts at levels <0.1 % BCR-ABL IS from 42 patients by (1) and (2) and 423 paired PB samples of 22 patients by (3) and (4). Two-proportion z-test was applied to compare the level of sensitivity.

Results: DNA analysis showed better score for BCR-ABL1 detection and quantification by qPCR (for scoring see Table 1) in 338/829 (41%) paired samples (PS) compared to mRNA, while BCR-ABL1 at mRNA level was better in 42 (5%) cases (p<0.0001). When we adjusted the level of sensitivity for mRNA and DNA analysis of PS, we found, that at level MR4-4.5 or 10-4 (n=484), respectively, DNA analysis was more sensitive in 163 (34%) PS, while the mRNA BCR-ABL1 was more sensitive in 27 (6%) PS (p<0.0001). At level MR5 and 10-5 (n=43), respectively, DNA was more sensitive in 14 (33%) PS and less sensitive than mRNA in 2 (5%) PS (p=0.0023). DNA analysis was more sensitive by ddPCR in 194/423 PS (46%), while mRNA was better in 6 PS (1.4%) (p<0.0001). DdPCR increased sensitivity of mRNA compared to qPCR in 96/614 PS (16%). We did not observe any differences in BCR-ABL1 detection between PB and BM using all 4 approaches in 7 patients with sustained DMR. In one patient BCR-ABL1 was positive by (3) and (4) in PB but not in BM. When we analyzed whole extracted DNA from BM by (4) we detected 2.2 BCR-ABL1 copies in 20x106 copies of ALB.