Ultra-deep sequencing speeds TKI resistance mutation detection

Haematologica 2016;101:830-838

medwireNews: Ultra-deep sequencing (UDS) can detect the T315I tyrosine kinase inhibitor (TKI) resistance mutation in patients with chronic myeloid leukaemia (CML) several months before conventional sequencing, study findings suggest.

“Although all low percentage (<15%) T315I mutations were detectable by Sanger sequencing at subsequent routine sampling time points, results from UDS would have allowed an earlier therapy intervention and earlier consideration of alloSCT [allogeneic stem cell transplantation], as recommended by the current [European LeukemiaNet] guidelines for patients with T315I mutations”, the study authors write.

“This would increase the time frame needed to prepare an alloSCT, e.g. initiation of HLA typing of patient’s siblings or the search for unrelated stem cell donors”, say Torsten Haferlach and co-workers, from the MLL Munich Leukemia Laboratory in Germany.

The 454 XL+ platform (Roche, Branford, Connecticut, USA) allows detection of the ABL1 tyrosine kinase domain mutation at loads of 1–2%, they explain, whereas conventional Sanger sequencing is only sensitive after 10–20% expansion of the mutated subclone.

Samples were examined from two cohorts of CML patients, the first including 40 patients who had failed sequential treatment with imatinib, dasatinib and nilotinib and had a known T315I mutation detected by Sanger sequencing on two to five occasions over a median of 5 months.

Backtracking of samples by UDS detected a T315I mutation level below 15% in 20 of these patients, the team reports in Haematologica.

The researchers say that differences between samples with and without a low T315I mutation level were not explained by sampling interval (median interval of 3 months for both) or by the time from initial CML diagnosis to T315I onset, with the mutation arising within a year of disease onset in 11 and 10 patients, respectively.

But the team found that it could take up to 14 years after diagnosis for the T315I mutation to arise, by which time patients were on a second- or third-line TKI.

Analysis of 21 patients from initial diagnosis to T315I detection showed that just two had achieved a major treatment milestone before developing the mutation and this was lost when the mutation arose.

The remaining patients failed to achieve a molecular or complete cytogenetic response before developing the T315I mutation, after a median of 7 months.

And the team notes that five of these 19 patients received TKI therapy for 12 months or longer despite not achieving a major molecular response (MMR) within this timeframe, a criterion for optimal response as per the European LeukemiaNet guidelines.

The researchers also found that patients who had a low level of mutations took a median of 3 months for the T315I to expand to greater than 15%.

Although none of these patients lost the T315I mutation during monitoring, the team admits that as the sample only included patients with at least one positive Sanger test, “we cannot exclude the possibility that low level mutations may arise but disappear before being detected by Sanger sequencing in a context of routine diagnostics”.

They therefore examined a second cohort of 42 patients who had achieved and lost an MMR and had no evidence of a resistance mutation on Sanger sequencing. But UDS failed to detect T315I mutation or other alterations associated with TKI resistance.

“In our study, a strong dominant outgrowth of the T315I clone over all other mutated clones was observed”, the team concludes.

“This demonstrated the strong resistance of the mutated cells against first- and second-line TKIs and the extraordinary importance of considering a T315I mutation before changing TKI.”

By Lynda Williams, Senior medwireNews Reporter

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