Analysis of the Sub-Clonal Origins of Compound Mutations in Patients with Refractory Ph+ Malignancies Treated with Ponatinib
Authors: Pritchard Justin R. , Schmitt Michael W. , Hodgson J. Graeme , Lustgarten Stephanie , Tang Shijie , Loeb Lawrence A. , Radich Jerald P. , Rivera Victor M. 
Published: November 2019

Background: Ponatinib is a tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL1 and all single resistance mutants. In the Phase 2 PACE study (NCT01207440), ponatinib induced high rates of major hematological response (MaHR) and major cytogenetic response (MCyR) in BP-CML (31% and 23%) and Ph+ ALL (41% and 47%) patients (pts), even though 91% of pts had received at least 2 prior TKIs. However, the median progression free survival was only 4 months for BP-CML and 3 months for Ph+ ALL (Cortes JE, et al. N Engl J Med 2013). Previous studies have shown that certain compound mutations can cause resistance to ponatinib (Zabriskie et al. Cancer Cell 2014), but such mutations were found to be extremely rare in CP-CML pts from the PACE trial despite being heavily pre-treated (Deininger MW, et al. Blood 2016). Here we utilize a multi-level sequencing strategy that combines Sanger Sequencing (SS), Next Generation Sequencing (NGS), and single molecule Duplex Sequencing (DS), which is orders of magnitude more sensitive than NGS, to profile the mutational mechanisms that may account for the survival outcomes observed in BP-CML/Ph+ ALL pts treated with ponatinib in PACE.

Methods: The BCR-ABL1 mutation status of the 267 CP-CML, 62 BP-CML, and 32 Ph+ ALL pts treated in the PACE trial was examined using SS and/or NGS as described previously (Cortes JE, et al. N Engl J Med 2013; Deininger MW, et al. Blood 2016). Mutations detected by SS at the end of treatment (EOT) but not at baseline (BL) were considered to have been gained during ponatinib treatment. DS was performed to determine whether mutations gained on treatment could be found in small sub-populations of cells at BL and to provide a direct measurement of the mutation burden in the ABL1 gene. Two distinct mathematical models were used to predict the size of pre-existing resistant sub-populations in BP-CML/Ph+ ALL pts based on the overall ABL1 mutation burden and the doubling time of clonal outgrowth.

Results: The number of BCR-ABL1 mutations in BP-CML/Ph+ ALL patients at BL, as detected by SS, was not a significant predictor of intrinsic resistance to ponatinib in refractory patients with Odds Ratios (OR) of 1.0 (0.2-4.9 95% CI) for MaHR and 2.4 (0.5-12.5 95% CI) for MCyR. However, a larger proportion of BP-CML/Ph+ ALL pts, 30/61, acquired mutations in BCR-ABL1 at EOT vs. 19/130 CP-CML pts (p<0.005, OR 7.7 [1.5-53.3 95% CI]). Nearly all BP-CML/Ph+ ALL, but not CP-CML, pts who acquired mutations had BCR-ABL1 mutations at BL detected by SS: 27/30 vs. 10/19 CP-CML pts (p=8.5E-7, OR 5.6 [2.7-12.1 95% CI]). At least 19/27 BP-CML/Ph+ ALL pts had compound mutations at EOT, including 2 pts with 3 mutations in the same allele. Since the presence of pre-existing mutations from earlier TKI therapy is associated with further gain of mutations on ponatinib treatment, we used ultra-sensitive DS to determine if these resistant clones could be detected at BL. In 3/18 pts tested, the resistant clone was directly detected at BL in a small minority of cells. Mathematical modeling of leukemia growth rates in pts with mutation gains predicts that pre-treatment resistant population sizes range from ~1 cell to 2.7 x 108 cells in the peripheral blood, out of a total population of leukemic cells that ranged from ~1010 to 5 x 1011. Furthermore, DS and mathematical modeling allowed us to predict that many, if not all, documented resistance mutations were pre-existing in most refractory BP-CML/Ph+ ALL pts treated with ponatinib in the PACE trial.

Conclusions: Clonal BL BCR-ABL1 mutation status determined by SS does not predict intrinsic resistance to ponatinib. However, in contrast to CP-CML, BP-CML/Ph+ ALL pts are more likely to relapse on ponatinib with acquired mutations in BCR-ABL1. Two distinct mathematical modeling strategies suggest that most, if not all, BCR-ABL1 resistance mutations in BP-CML/Ph+ ALL pts that emerge during ponatinib treatment were pre-existing. The majority of these pre-existing mutations were compound mutations whose evolutionary path was likely determined by prior TKI therapy. These findings emphasize the risk of the large population expansions that occur following prior TKI treatment failure and may explain the relatively high 2-yr event free survival rate of 81% observed in previously untreated Ph+ ALL pts treated with ponatinib (+hyper-CVAD) (Jabbour E, et al. Lancet Oncol 2015, Sasaki, et al. Cancer 2016).

Study sponsor: ARIAD Pharmaceuticals, Inc.

Disclosures

Pritchard: ARIAD: Employment, Equity Ownership. Schmitt:TwinStrand: Equity Ownership. Hodgson:ARIAD: Employment, Equity Ownership. Lustgarten:ARIAD: Employment, Equity Ownership. Tang:ARIAD: Employment, Equity Ownership. Loeb:TwinStrand: Equity Ownership. Radich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; Pfizer: Consultancy; ARIAD: Consultancy; TwinStrand: Consultancy. Rivera:ARIAD: Employment, Equity Ownership.