ALBERT

Description: Abstract 2549 Background: BCR-ABL1 mutation testing is recommended for CML and Ph+ ALL patients who fail first line tyrosine kinase inhibitor (TKI) therapy or who have a suboptimal response to therapy. BCR-ABL1 mutations in the kinase domain (KD) of ABL1 account for at least 40–50% of all TKI resistant cases. Rare mutations such as E123Q and T212R in the regulatory domain of ABL upstream of the kinase domain have also been reported to lead to resistance to imatinib. The current gold standard for BCR-ABL1 mutation detection is Sanger sequencing, which has an analytical sensitivity of ∼10–30%. Based on recent findings that mass spectrometry can identify low level BCR-ABL1 mutations that confer clinical resistance in patients sooner than Sanger sequencing, it is likely useful to have a significantly more sensitive BCR-ABL1 test than Sanger sequencing. Although commercial NGS cancer panels have included ABL1 in the region of interest, ABL1 resistance mutations should be sequenced from BCR-ABL1 fusion transcripts instead of being sequenced from genomic DNA as in the commercial panels. Here we developed a fusion transcript based BCR-ABL1 mutation assay on the scalable and cost-effective Ion Torrent platform that has 1–5% sensitivity and comprehensive coverage of the kinase domain, regulatory domain, and the SH2/SH3 domains. The assay was designed to detect both the major and minor BCR-ABL1 fusion gene products and can also detect the micro BCR-ABL1 fusion product accounting for over 99% of all CML and Ph+ ALL patients. Methods: RT and long range PCR was performed to amplify BCR-ABL1 e1, e13, and e14 fusion transcripts and the PCR products were enzymatically fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with OneTouch system and sequenced with Ion Torrent PGM. Sequencing data was analyzed with Torrent Suite 2.2 and the associated variant caller with variant frequency cutoff adjusted to 1%. Results: Initial work with cell lines harboring the T315I mutation in both e1 and e14 BCR-ABL1 transcript types diluted into wild type cell line demonstrated that Ion Torrent NGS can detect T315I at least down to 1%. In a set of 17 blinded clinical samples, Ion Torrent NGS not only identified all the mutations found by Sanger sequencing but additionally identified rare imatinib resistant mutations such as K357E (MMD-3) present at 7.6%; this mutation was previously reported in patient CD34+CD38-stem cells (Table 1). This patient also expressed the E255K resistance mutation; in patients, the presence of multiple resistance mutations has been shown to be an important predictor of poor response. Understanding whether compound mutations are present in cis or in trans may be important in understanding therapy resistance. For patient MMD-9, although both the predominant mutation G250E (79%) and subclone E255V(12.6%) were identified by both Sanger sequencing and NGS, only Ion Torrent was able to show that the two mutations were on different reads, indicating that the mutations are on different alleles. The BCR-ABL1 Ion Torrent based assay reads DNA fragments between 150–200bps in size and can identify cis and trans mutations from individual fragments with this read length, which is not possible by Sanger sequencing. In MMD-9, another low frequency subclone F359C (3%) was detected by only NGS and may have important implications due to its reported sensitivity to dasatinib and not imatinib or nilotinib. Similarly, in MMD-10 the E255V predominant mutation was identified by both NGS and Sanger sequencing, while the F317L mutation with a low frequency of 1.70% was solely detected by NGS. In this patient, a combination of dasatinib and nilotinib treatment may be required to eliminate the dasatinib sensitive E255V dominant clone and F317L nilotinib sensitive subclone. Ion Torrent NGS was also capable of identifying and calling the 35 base pair 475 insertion from 4 samples (MMD-2 and 8–10), only two of which were detected by Sanger sequencing. In our ongoing study, the low-level mutations not detected in Sanger sequencing will be confirmed with analyses on the MiSeq platform and mutation enrichment methods. Additional patient samples representing 30 CML patients will be analyzed and presented. Disclosures: Wong: MolecularMD: Equity Ownership.

Description: Background Many CML patients treated with tyrosine kinase inhibitors (TKIs) eventually develop resistance as a result of ABL1 kinase domain (KD) mutations, and sequential treatment with different TKIs may select for multiple BCR-ABL1 mutations. Whether multiple mutations arise in distinct clones (in trans, or polyclonal mutations) or instead are present within the same BCR-ABL1 molecule (in cis, or compound mutations), has been shown to have important implications with respect to TKI sensitivities (Eide, C.A. et al., Blood 2011). Distinguishing between polyclonal and compound mutations, or mutation phasing, for the ABL1 KD has not been clinically practical with standard mutation detection methods. Here we have developed a highly sensitive next-generation sequencing (NGS) assay on the Ion Torrent PGM along with a proprietary data analysis pipeline that together enable deep sequencing of the BCR-ABL1 KD and neighboring domains with a 1% limit of detection and quantitative reporting of mutation phasing. Methods RT and long range PCR was performed to amplify BCR-ABL1 e1a2/3, e13a2/3, and e14a2/3 fusion transcripts and the PCR products were enzymatically randomly fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with the OneTouch system and sequenced with the Ion Torrent PGM using 400 bp sequencing chemistry. Sequencing data were analyzed with Torrent Suite 3.4.2 with variant frequency cutoff adjusted to 1%. Variants were further annotated with a proprietary analysis pipeline and variant report was produced after manually reviewing variants by Integrative Genomics Viewer. If more than one non-synonymous variant was reported in a sample, a proprietary phasing analysis pipeline was applied to report the mutation spectrum of all of the combinations of multiple mutations in the sample. Results To validate the accuracy of the sequencing method which employs 400 bp sequencing chemistry, we compared this assay with our previously validated BCR-ABL1 NGS assay based on Ion Torrent 200 bp sequencing chemistry for a set of clinical specimens from CML patients previously treated with TKI. Results were highly concordant and similarly sensitive, with 11/11 variants (frequencies ranging from 2% to 100%) identified with comparable frequencies by both methods. To evaluate the specificity of the phasing analysis, an artificial sample was created by mixing two samples (b2_10 and b2_6) with 6 distinct variants present at ratio of 1:19. Because variants are unique in each sample, any compound mutation composed of b2_10 variant and b2_6 variant identified would be false positive. The false positive error rate (percentage of b2_6 variant as compound mutation with b2_10 variant and vice versa) ranged from 0 – 0.6%, which was consistent with sequencing error rate. We conservatively define a compound mutation as true if it is present in at least 5% of any one of the component variants in the compound mutation. Mutation detection and phasing analysis were reproducible on different chips (314 v2 and 318 v2) and different library preps from the same long range PCR product of BCR-ABL1. Table 1 shows the mutation spectrum from sample b2_6. Of the four variants detected, L248V and G250E were mutually exclusive (in trans), while T315I and M351T were present as compound mutations with each other and, separately, with either L248V or G250E. Notably, 〉86% of the molecules harbored single mutations, and no compound mutations containing more than 2 variants were observed. Conclusions We have developed and validated a sensitive NGS assay that enables deep sequencing of the BCR-ABL1 KD and neighboring domains along with quantitative mutational phasing. This method has been applied in evaluating 〉250 clinical specimens for a clinical trial of a third-generation TKI(results reported separately). The ability to easily determine the mutation phasing of a CML patients’ mutation profile using this assay will allow for investigations into compound mutation-based resistance mechanisms and may be used to better guide treatment decisions. Disclosures: Li: MolecularMD: Employment. Yan:MolecularMD: Employment. Darwanto:MolecularMD: Employment. Fang:MolecularMD: Employment. Liu:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Toplin:MolecularMD: Employment. Spittle:MolecularMD: Employment. Galderisi:MolecularMD: Employment.

Description: In Ph+ ALL, the absence of detectable disease has shown prognostic value for a reduced risk of relapse and improved survival. However, as the level of undetectable disease is determined by the lower limit of detection of the test in use, standardization of such an endpoint for a drug regulatory submission is critical. To date, no tyrosine kinase inhibitor (TKI) has received approval for the newly diagnosed Ph+ ALL adult patient population in the US. Takeda (Millennium Pharmaceuticals, Inc.) is conducting a phase 3, randomized, open-label, multicenter efficacy study comparing ponatinib versus imatinib, administered in combination with reduced-intensity chemotherapy, in participants with newly diagnosed Ph+ ALL (NCT03589326). The primary endpoint for this study is minimal residual disease (MRD)-negative complete remission (CR), where MRD-negativity is defined as a BCR-ABL:ABL raw ratio of ≤0.01% (MR4.0) in bone marrow aspirate samples at the end of induction. Patients who achieve post-induction ponatinib or imatinib maintained MRD-negative CR will potentially delay or avoid stem cell transplantation. Previously, for the purposes of initiating and monitoring treatment free remission or discontinuation of TKI therapy in chronic phase CML patients, we developed and validated the MRDx® BCR-ABL Test which is an FDA authorized test for the quantitative detection of BCR-ABL e13a2 or e14a2 transcripts. This test will be used in this study and reports BCR-ABL:ABL levels on the International Scale (IS) with traceability to the World Health Organization (WHO) first International Genetic Reference Panel and with a limit of detection below 0.0032% (i.e., MR4.5). Similarily, for assessment of the e1a2 (p190) BCR-ABL:ABL transcripts, we developed and validated a one-step reverse transcription, quantitative polymerase chain reaction (RT-qPCR) test in order to accurately and precisely assess all clinical decision points and disease levels for this study. Because of the lack of available reference material for e1a2, a droplet digital PCR (ddPCR) based test was co-developed to quantify e1a2 BCR-ABL copy numbers in bone marrow aspirates, as well as in peripheral blood samples (to allow assessment of concordance). e1a2 in vitro transcribed RNA calibrators assign copy numbers to determine the e1a2 BCR-ABL:ABL raw % ratios of unknown samples. The e1a2 RT-qPCR test exceeded an analytical sensitivity of MR4.5 (0.0032% raw ratio of BCR-ABL:ABL) with a dynamic linear range from MR4.5 to MR1.0. The test also includes cell line derived RNA assay controls formulated to 10%, 0.1% and 0.01% BCR-ABL:ABL, necessary for decision points in the clinical trial. Validation studies included limit of blank, limit of detection (LOD), limit of quantification, assay range, analytical specificity, repeatability, reproducibility (multi-day, multi-operator, and multi-instrument), and accuracy by comparison to a reference method (ddPCR). The validation of the e1a2 RT-qPCR test with bone marrow aspirate samples was conducted with 1 µg RNA inputs per well and LOD was also verified with 0.5 µg RNA input per well. In conclusion, the validated e1a2 RT-qPCR test allows for accurate standardization of BCR-ABL:ABL measurement across multiple centers in an international Phase 3 study. The e1a2 RT-qPCR test data will be used to assess the primary endpoint in the first registrational trial to be conducted in newly diagnosed Ph+ ALL adult patients. Disclosures Drafahl: MolecularMD, Corp: Employment. Smith:MolecularMD, Corp: Employment. Graham:MolecularMD, Corp: Employment. Glynn:MolecularMD, Corp: Employment. Spittle:MolecularMD, Corp: Employment. Verrow:Takeda (Millennium Pharmaceuticals, Inc.): Employment. Rivera:Takeda (Millennium Pharmaceuticals, Inc.): Consultancy. Srivastava:Takeda (Millennium Pharmaceuticals, Inc.): Employment. Hawkins:MolecularMD, Corp: Employment. Galderisi:MolecularMD, Corp: Employment, Equity Ownership.