ALBERT

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.

Description: Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKI) has led to progressively lower levels of disease burden and higher rates of complete molecular responses. Very few assays with an analytical sensitivity of 4.5-logs (0.00316% BCR-ABL/ABL) have been validated, and assay standardization at these levels has been problematic (EUTOS, 2007). In order to have an International Scale (IS) standardized assay that can precisely and accurately detect 4.5 log reductions in BCR-ABL/ABL ratios, the test system design must be tightly controlled to ensure this critical performance metric can be reproducibly met. The MolecularMD MRDxTM BCR-ABL Test was developed and validated to be an accurate, reproducible, and highly sensitive IS-standardized solution to measure minimal residual disease (MRD) in CML patients using either PAXgene Blood RNA or EDTA blood collection tubes. The MRDx Test is a one-step quantitative real-time polymerase chain reaction (RT-qPCR) test that allows for quantitation of BCR-ABL e13/14a2 (b2/3a2) transcripts (covering ≥ 95% of CML patients) and ABL transcripts in RNA extracted from peripheral blood. To achieve a sensitivity of 4.5 logs, several one step enzyme systems were evaluated against RNA samples that spanned the potential dynamic range of the assay (MR1.0 to MR5.0: MR1.0=1 log molecular response (MR), or 10% IS; MR5.0=5 log MR, or 0.001% IS). The one step enzyme system with the best sensitivity and precision was chosen based on the performance of three distinct lots. In addition, in order to better control for the variation of the reverse transcription, in vitro transcribed RNA calibrators were developed to create a standard curve for copy number determination rather than the more commonly used plasmid calibrators that may not accurately reflect the same processing as the patient sample RNA. The limit of detection (LOD) for blood drawn into PAXgene Blood RNA tubes was validated by testing a dilution series created from a baseline CML patient blood sample diluted into non-diseased subject blood. Creating this type of dilution series allows for the determination of the LOD in the actual clinical sample matrix as compared using cell line dilution series that have much higher BCR-ABL and ABL copy numbers than routine patient specimens. Based on the analysis of each level of sample tested over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.7 (Mean=MR4.9, 95% CI=MR5.2 to MR4.7) using the more conservative upper bound of the 95% confidence interval. The precision was evaluated based on the standard deviation of the log10 BCR-ABL/ABL ratio and was found to be ≤ 0.20 SD at MR5.0 and above. The LOD for blood drawn into EDTA blood collection tubes was validated by creation of a dilution series from a baseline CML patient RNA sample diluted into RNA isolated from blood of non-diseased subjects. Based on the number of samples with detectable BCR-ABL in at least 95% of replicates over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.9 (Mean=MR5.0, 95% CI=MR5.1 to MR4.9) using the more conservative upper bound of the 95% confidence interval with precision being ≤ 0.22 SD at MR5.0 and above. The accuracy of the BCR-ABL copy numbers, ABL copy numbers, and BCR-ABL/ABL ratio using the MRDx BCR-ABL Test was evaluated for ten patient samples using one step droplet digital PCR (ddPCR) as a reference method. For the BCR-ABL copy numbers, ABL copy numbers, and the BCR-ABL/ABL ratio, the bias of the MRDx Test relative to ddPCR by Bland-Altman method was 0.053, 0.030, and 0.023 respectively. The MRDx BCR-ABL Test reports on the IS with a conversion factor of 1.0 by use of the WHO International Standard 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR; secondary standards were created for bi-annual monitoring of the assay conversion factor to ensure accurate reporting of patient results on the IS. Confirmation of the conversion factor was done by an independent laboratory using different real-time PCR instruments and multiple technicians. Based on the validation data, the MRDx BCR-ABL Test is an accurate, reproducible, and highly sensitive IS-standardized solution to the growing need for a reliable and robust quantitative BCR-ABL assay that can be used for the monitoring of minimal residual disease in CML patients. Disclosures: Toplin: MolecularMD: Employment. Drafahl:MolecularMD: Employment. Eibl:MolecularMD: Employment. Fjeld:MolecularMD: Employment. Yager:MolecularMD: Employment. McAdams:MolecularMD: Employment. Hawkins:MolecularMD: Employment. Galderisi:MolecularMD: Employment.