Ultra-Deep Sequencing Leads to Earlier and More Sensitive Detection of the TKI Resistance Mutation p.T315I in CML

Background: Chronic myeloid leukemia (CML) cells can acquire resistance to tyrosine kinase inhibitors (TKI) that in ~40% of cases is due to acquisition of mutations in the ABL1 kinase domain of the BCR-ABL1 transcript. The p.T315I (c.944C>T) mutation (mut) mediates resistance to most BCR-ABL1 TKIs (Imatinib, Dasatinib, Nilotinib and Bosutinib), whereas sensitivity to ponatinib has been demonstrated. Patients with p.T315Imut show a rapid increase in malignant cell burden and can progress to blast crisis. An earlier detection of the p.T315Imut may allow TKI treatment intervention ahead of disease progression. However, the sensitivity of conventional Sanger sequencing for detection of mutations is not less than 10-20%.

Aim: To study the dynamics of evolution and progression of the p.T315Imut using ultra-deep sequencing (UDS) in comparison with Sanger sequencing.

Patients and Methods: We selected 18 CML patients with high p.T315Imut levels originally detected by Sanger sequencing for routine diagnostics. Subsequently, we backtracked prior blood samples of all patients for a mean period of eight months (2-15 months) before detection of p.T315Imut by Sanger sequencing, analyzing 3-7 time points per patient. Patients (4 female and 14 male) had a median age of 60 years (18-84 years) and received treatment as follows: only Imatinib (n=3), only Nilotinib (n=3), only Dasatinib (n=1), treated with two prior (n=6) or three prior TKIs (n=5) by the time of p.T315Imut detection by Sanger sequencing. For more sensitive mutation detection, we amplified the BCR-ABL1 fusion transcript and designed two sequencing amplicons (550 bp and 575 bp) for UDS with the XL+ Kit for extended read length (Roche/454, Branford, CT). A minimal read coverage of 1,000 per base was reached. Our backtracking study by UDS was performed on samples sent in at intervals of approximately 3 months.

Results: To prove high sensitivity of UDS with the 454 XL+ protocol we performed dilution experiments for three sequence variants and replicated sequencing experiments with low level mutations. The detection limit was at 1-2% mutation level and thus is 10-fold better than the sensitivity reached by Sanger sequencing. At the time point of initial routine diagnosis of p.T315Imut the median mutation load was 87.5% (30-100%) by Sanger sequencing and very similar by UDS (median: 84%; range: 40-99%; R²=0.7). In 6/18 patients backtracking identified a sample with a low p.T315I mutation level of <5% (1.9-13.6 months, median: 3.2 months) before a mutation load of >10% (Sanger sequencing detection level) was reached. Thus, in 33.3% of all cases a small, early clone of CML with p.T315Imut was identified. At subsequent time points, all 6 patients experienced a strong increase of the p.T315Imut level (>50%), which represents the very fast expansion of the mutated clone. In a second subset of 10 patients, the p.T315Imut load was already >30% when first detected by UDS. The median interval to the last p.T315I negative time point was 2.4 months (0.9-3.5) and no sample between the p.T315I negativity and high positivity was available. This subset confirms the fast outgrowth of the p.T315Imut positive clone. The p.T315Imut load had a median increase of 0.9% (0.2-3.1%) per day, when calculated as average increase from the last negative sample to the time point with maximum mutation load. The other 2 patients had high p.T315Imut levels (>40%) for our entire monitoring period.

At the time of p.T315I detection by UDS, we observed eight patients with additional resistance mutations. The accumulation of mutations in one clone results in an extremely resistant CML. This was detected in one patient, where a p.T253H clone (Imatinib and Dasatinib resistant) gained the p.T315Imut. This clone expanded to 73% within 79 days. In contrast, we identified five cases with multiple CML clones carrying different mutations. However, the p.T315Imut clone was able to overgrow up to six other resistant clones.

Conclusions: We showed: 1) the p.T315Imut rapidly increases upon occurrence, supporting the relevance of regular mutation monitoring in CML patients, when resistance to TKIs is suspected. 2) that small p.T315Imut clones in the 1-2% range can be sensitively detected by UDS in 33% of all samples if sampling intervals are within the 3 months range. 3) earlier detection of the p.T315Imut by UDS is a potentially valid method to allow a prompt change of TKIs before clonal expansion of the p.T315Imut cells.