Detection of Actionable BCR-ABL1 Kinase Domain (KD) Mutations in Chronic Myeloid Leukemia (CML) Patients with Failure and Warning Response to Tyrosine Kinase Inhibitors (TKIs): Potential Impact of Next-Generation Sequencing (NGS) and Droplet Digital PCR (ddPCR) on Clinical Decision Making
Authors: Soverini Simona , Martelli Margherita , Bavaro Luana , De Benedittis Caterina , Iurlo Alessandra , Galimberti Sara , Pregno Patrizia , Bonifacio Massimiliano , Lunghi Francesca , Castagnetti Fausto , Gugliotta Gabriele , Rosti Gianantonio , Tiribelli Mario , Velotta Vanessa , Stagno Fabio , Russo Rossi Antonella , D'Adda Mariella , Baratè Claudia , Crugnola Monica , Luciano Luigiana , Laginestra Maria Antonella , Corner Adam S , Maar Dianna , Percesepe Antonio , Pileri Stefano , Pane Fabrizio , Saglio Giuseppe , Baccarani Michele , Martinelli Giovanni , Cavo Michele 
Published: February 2020

A variety of mechanisms underlie the lack or loss of response to TKIs in patients (pts) with CML, but acquisition of point mutations in the BCR-ABL1 KD is, at present, the only actionable one. Detection of a TKI-resistant mutation by Sanger Sequencing (seq) should trigger a change of therapy and, in some cases, guide TKI selection. Although research studies have shown that NGS may hold added value over Sanger seq for BCR-ABL1 KD mutation screening, routine NGS is technically demanding and expensive and is not yet widely available. Newer methods, like ddPCR, might represent an attractive alternative. Here we set out to 1) assess the actionability of results, hence the potential clinical benefit, of more sensitive NGS-based testing vs Sanger seq in a consecutive series of CML pts who had non-optimal response to TKI therapy according to the 2013 ELN recommendations, and 2) test a novel ddPCR-based multiplex assay for rapid screening for a panel of BCR-ABL1 KD mutations relevant for TKI selection.

Between January 2015 and May 2019, samples from 712 CML pts followed at one of 66 GIMEMA CML Working Party hematology centers were referred to our laboratory for BCR-ABL1 KD mutation testing because of a Failure (n=251 pts) or Warning (n=461 pts) response to TKI therapy. In parallel to Sanger seq, NGS of amplicons generated by nested reverse transcription(RT)-PCR was performed on a Roche GS Junior instrument until April 2017, and on an Illumina MiSeq instrument from May 2017 on. Read alignment and variant calling was done using AmpSuite software (SmartSeq). Variants detected in &lt;3% of BCR-ABL1 transcripts were filtered out. ddPCR was performed on a QX200 instrument using a single-tube assay (BioRad) containing primers and probes for the simultaneous detection and discrimination of 2nd generation TKI (2GTKI)-resistant mutations: T315I/A, Y253H, E255K/V, F359V/I/C, V299L and F317L/V/I/C. Data analysis was performed using QuantaSoft software.

Among Failures, pts positive for BCR-ABL1 KD mutations by Sanger seq were 88/251 (35%). NGS detected low level mutations in 38/251 (15%) additional pts who were negative for mutations by Sanger seq. Moreover, 31/251 (12%) Failure pts were found to have low level mutations additional to those detectable by Sanger seq. Among low level mutations detected by NGS, those actually relevant for TKI selection (2GTKI-resistant) were identified in 22/251 (9%) pts. Compound mutations were found in 10 pts (4%; all progressed to blastic phase). The average turnaround time (TAT) of routine NGS-based analysis was 15 working days (range, 11-32). Thus, in the setting of Failure, NGS-based mutation scanning of the whole KD is of real clinical benefit in a minority of pts while increasing the TAT.

Among Warnings, pts positive for BCR-ABL1 KD mutations by Sanger seq were 65/461 (14%). NGS detected low level mutations in 97/461 (21%) additional pts who were negative for mutations by Sanger seq. All Warning pts positive for low level resistant mutations who were not switched to another TKI turned their response into Failure after 3 to 8 months.

The multiplex digital PCR assay proved capable to accurately identify and separate the T315I/A, F317L/V/I/C, Y253H, E255K, F359V/I/C, E255V, V299L mutations in five spatially distinct clusters (pan-resistant, Dasatinib-resistant, Nilotinib-resistant, Nilotinib- and Bosutinib-resistant, Dasatinib- and Bosutinib-resistant, respectively) starting from as little as 30ng of total cDNA or 1ng of the 1.7kb 1st PCR product. Very good concordance was observed between ddPCR- and NGS-identified mutations irrespective of mutation frequency or cluster proximity, even for compound mutations. Extensive assay assessment will be presented. TAT of ddPCR was 1 day.

In conclusion:

- Mutation testing in the Failure setting aims to a timely and rational TKI switch. The incidence of low level mutations relevant to the selection of subsequent-line therapy and the TAT of routine NGS in our cohort suggest that multiplex ddPCR would be an easier and faster alternative.

- Mutation testing in the Warning setting may identify pts who need a change in therapy rather than a 'watch and wait' approach. In our cohort, approx 1/5 of the Warning pts negative by Sanger seq had low level mutations resistant to the TKI they were receiving, that ultimately led to Failure. Earlier detection of emerging resistant mutations enabled by NGS (or by ddPCR in pts receiving 2GTKIs) should support proactive TKI switch.

Disclosures

Soverini: Incyte: Consultancy. Iurlo:Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Bonifacio:BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Castagnetti:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tiribelli:Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Stagno:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; BMS: Honoraria. D'Adda:Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Crugnola:Incyte: Honoraria; Novartis: Honoraria. Corner:BioRad: Employment. Maar:BioRad: Employment. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Saglio:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Martinelli:Pfizer: Consultancy; ARIAD: Consultancy; Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy. Cavo:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.