Low Level BCR-ABL Mutations Below the Detection Limit of Current Standard Screening Techniques Occur Predominantly in the CD34+ Progenitor Cell Compartment in Chronic Phase CML Patients At Diagnosis. A Substudy of the ENEST1st Trial

Abstract 1671

The presence of BCR-ABL kinase domain mutations below the detection limit of conventional screening techniques (low level mutations, LLM) predicts outcome of subsequent therapy in patients with imatinib resistance (Parker et. al JCO 2011 and Blood 2012). We have further evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of complete molecular responses after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). Here, we have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment in comparison to total white cells (TWBC).

Sixty nine ENEST1st study patients with CP CML provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34⁺ selection was carried out by MACS^® (Miltenyi Biotec) and the CD34⁺ purity was subsequently determined by fluorescent activated cell sorting (FACS). The results were compared to those derived from stored TWBC from 23 of the same patients and a further 16 patients at diagnosis.

Aliquots of 10⁵ CD34⁺ or at least 10⁶ TWBC were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3–3.5 log). No patients showed BCR-ABL kinase domain mutations detected by Sanger sequencing spanning ABL exons 4–9. Forty five of 69 patients (65%) with 10⁵ CD34⁺ cells and a documented CD34⁺ purity of >50% were available for BCR-ABL amplification. Amplification was successful from 36 (52%) of these CD34⁺ samples and from 38 of the 39 (97%) TWBC samples.

A total of 180 L-PCR assays of CD34⁺ cells identified 29 (16%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) in CD34⁺ cells from 21/36 patients (58%). In comparison, 190 assays of TWBC identified 10 (5%) mutations (T315Ix3, Y253Hx6, E255Vx1, p=0.0005) in 8/38 patients (21%, p=0.001 Fishers exact test). Significantly more T315I (33%) and E255K (22%) mutations were observed in CD34+ cells than in TWBC (8%, p=0.007 and 0% p= 0.003 respectively). The quantitative levels of all mutant alleles were median 0.135 (range 0.06–0.535) and 0.1 (range 0.04-0, 25) BCR-ABL^mutant/ BCR-ABL^unmutated for mutations in CD34⁺ cells and TWBC, respectively and were not significantly different.

Where both CD34+ and TWBC were available from the same patient (n=23), 11 patients showed a total of 18 mutations in the CD34⁺ fraction but only one of these mutations was confirmed in TWBC. One additional mutation was detectable in the TWBC. The remaining 12 patients with no detectable mutation in the CD34⁺ fraction showed 3 mutations (2x Y253H, T315I) in 2 patients in TWBC only.

In conclusion, LLM with either no (T315I) or intermediate (Y253H, E255K/V, F359V) sensitivity to nilotinib are detectable in CP CML patients at a frequency of 21% in the TWBC but with a significantly higher frequency of 58% in the enriched CD34⁺ progenitor cell compartment. Longterm patient follow up on the ENEST1st and ENESTobserve studies will allow analysis of the relationship between LLM and clinical outcomes on nilotinib.