Clinical Intervention Using NGS-Based Kinase Domain Mutation Testing in CML Patients
Authors: Kizilors Aytug , Harrington Patrick , Jackson Sophie , Lea Nicholas , Newman Joel , Gale Richard , Hassan Sandra , Dimitriadou Evangelia , Crisà Elena , Ficinski Luke , Sa Isabel , Arasaretnam Anita , Dhanapal Vijayavalli , Corbett Timothy , Ireland Robin M. , Pagliuca Antonio , Mufti Ghulam J. , De Lavallade Hugues 
Published: October 2019

Abstract
Kinase domain (KD) mutations in the BCR-ABL1 gene are associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). Next-generation Sequencing (NGS) allows detection of low-level KD mutations (<20% VAF), although the detection of true low level KD mutation can be challenging. We have previously reported an improved single round PCR based NGS method to ensure higher accuracy for clinical use (Kizilors et al. ASH 2015). To implement this improved assay in our routine clinical setting, we obtained ISO 15189 accreditation for NGS based BCR-ABL1 KD mutation detection.
We have previously shown the clinical significance of low-level KD mutation by NGS. However our study, along with previous studies, was retrospective. Since August 2014, we have been using NGS-based assay (in replacement of Sanger sequencing) for clinical intervention, allowing us to study the clinical relevance of low level mutations and monitor the kinetics of mutated clones.
We herein report the outcome of patients in whom clinical intervention was made based on NGS KD mutation screening and monitoring.
We analyzed the outcome of 340 consecutive Chronic Phase CML patients treated with first-line TKI at our institution and within our network (imatinib 235, nilotinib 49, dasatinib 10, unknown 46), of whom 70.42% achieved CCyR with first-line TKI. NGS-based KD mutation testing was done retrospectively until January 2014, and prospectively from that date.
Overall, a mutation was found in 41(12.05%) patients at a median time of 14 months (range, diagnosis to 156 months). Twenty five different mutations were identified, including T315I (n=9), M244V (n=8), F317L (n=8) and E255K (n=6). Of 41 patients with mutated clone, 27 were found to have a mutation conferring resistance to the ongoing TKI treatment (defined as 'clinically relevant' mutation). In 8 patients, the mutation was sensitive to the TKI treatment received (M244V in 6 patients, M351T in one patient on imatinib and an E255V in one patient on dasatinib), two patients with intermediate resistance to the treatment received (H396R and E355G) and four patients with mutations of unknown clinical significance (Q252R, E450K and E450G). Of the 27 patients with clinically relevant mutations, 8 patients were part of our initial retrospective NGS screening study, of whom 3 had clinical intervention based on Sanger Sequencing results.
From August 2014, clinical intervention was made based on KD mutations found by NGS. Eighteen patients had their TKI treatment changed because of mutations found to confer resistance to their ongoing TKI (including 2 with unknown clinical significance), of whom 14 had single mutation (F317L, n=3; T315I, n=2; Y253H, n=1; F359V, n=1; L248V, n=1; G250E, n=1; F311L, n=1; E459G, n=1; E450K, n=1; F359I, n=1; E450G, n=1). Four patients had multiple mutations, including two patients who developed mutations sequentially.
Of the 18 patients who had clinical intervention, the first detection of the KD mutation was at a low-level in 13 patients.
Response to change of TKI was seen in 12 patients, of whom 11 achieved a stable MR3, and one patient with a 2.2 log reduction in BCR-ABL transcript levels. One patient proceeded to allogeneic HSCT and four patients did not show significant response at last follow up. Finally, one patient had a low level Y253H mutation detected while on nilotinib which initially reduced after TKI change and then subsequently increased while gaining an additional T315I at the time of Blastic Phase transformation. Response to combining chemotherapy and ponatinib was seen with the achievement of sustained MR3 until allogeneic HSCT.
Kinetics of mutated clone(s) was monitored post TKI change and all patients who responded experienced a reduction in their clone size. Among 12 patients who responded to TKI change, 3 had multiple mutations. One patient had a low level E255K mutation at 4% at 3 months despite achieving CCyR on nilotinib and subsequently developed an additional T315I mutation after 15 months upon loss of CCyR. This patient achieved a sustained MR3 upon switch to ponatinib with concomitant drop in the T315I clone size.
To our knowledge, this is the first report of clinical intervention using NGS based KD mutation detection in the management of CML patients. Our findings suggest that NGS may improve clinical decision-making in these patients. A prospective clinical study using NGS (acronym CALLS) is currently ongoing in the UK.

Disclosures
Kizilors: Incyte: Honoraria, Research Funding. Harrington:BMS: Research Funding. Pagliuca:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gentium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Mufti:Celgene: Research Funding. De Lavallade:Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding.