ALBERT

Description: Abstract 917 Background: Secondary kinase domain (KD) mutations represent the most well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In CML, multiple TKIs with different mutation profiles are approved and the ability to detect KD mutations at the time of disease progression can impact therapy choice. To optimize clinical impact, second generation TKI selection must consider the majority TKI-resistant mutant population as well as smaller mutant sub-populations that may be selected with subsequent treatment. Sequential TKI therapy is associated with additional complexity: multiple mutations can coexist separately in an individual patient (“polyclonality”) or can occur in tandem on a single allele (“compound mutations”). Multiple mutations are associated with poor clinical outcome (Parker et al., Blood 2012). Compound mutations can cause in vitro resistance to ponatinib, the only TKI clinically active against the highly resistant T315I mutation (Eide, et. al, ASH 2012 abstract #1416). Currently, no clinically adaptable technology can distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD, most next-generation sequencing platforms cannot generate reads of sufficient length to determine if mutations separated by ≥500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) sequencing technology is a third generation deep sequencing technology capable of achieving average read lengths of ∼1000bp and frequently 〉3000bp, enabling sensitive and accurate sequencing of the entire ABL KD on a single strand of DNA. Though allele-specific detection methods such as MassARRAY offer sensitivity as low as ∼0.5%, these assays are designed to detect a limited number (∼31) of mutations whereas SMRT sequencing offers an unbiased approach capable of detecting novel variants. We sought to (1) develop a potential clinically-applicable SMRT sequencing assay for the detection of BCR-ABLKD mutations capable of distinguishing polyclonal and compound mutations, and (2) compare the accuracy and sensitivity of this method to standard sequencing and MassARRAY. Results: We assessed 54 samples from 36 CML patients who had clinically relapsed on ABL kinase inhibitor therapy and were previously analyzed by standard sequencing, and in a subset, by MassARRAY. We amplified an 863bp area of the BCR-ABLKD from patient-derived cDNA with primers containing 5' barcodes, enabling sequencing of 6 to 8 patient samples on a single SMRT cell on a single run. On average, 2519 reads were obtained for each sample per run (range 330 to 10,240). All of 131 known mutations detected by MassARRAY were identified by SMRT sequencing using a p-value threshold of 1.03e–03. SMRT sequencing also identified all 107 known mutations detected by direct sequencing with a p-value threshold of 6.0e–08. In addition to these known mutations, SMRT sequencing detected an additional 1320 non-silent mutations across all patient samples using a strict p-value threshold cut-off of 6e–08, ranging in abundance from 0.2% to 17% (median 0.75%). Among 47 samples where 〉1 mutation was detectable by direct sequencing or MassARRAY, SMRT sequencing revealed that 40 (85%) had compound mutations detectable at a frequency of ≥1. In total, we detected 73 different compound mutations at a frequency of ≥1%. In all cases where compound mutations were detected and more than one treatment timepoint was available, at least one compound mutation clearly evolved from a mutation detectable at a prior timepoint. In the most complex case, 4 separate mutations yielded 8 different mutant alleles. Conclusions: Pacific Biosciences RS SMRT sequencing detects KD mutations in patient samples with sensitivity comparable to or better than MassARRAY and can distinguish compound from polyclonal mutant clones. Among patient samples with multiple mutations, compound mutations were detectable in the vast majority of samples by SMRT sequencing, revealing a complex mutational landscape not demonstrable by other clinically viable sequencing methods and previously unappreciated. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles by SMRT sequencing promises to further facilitate a personalized approach to patient management and inform models of disease evolution. Disclosures: Brown: Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Branford:Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid : Consultancy. Shah:ARIAD, Bristol Myers-Squibb: Consultancy, Research Funding; Novartis: Consultancy.

Description: MHC class I (MHC I) is essential to NK- and T-cell effector and surveillance functions. However, it is unknown whether MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I Dk, a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of murine (M)CMV infection. Here we assessed the significance of NK inhibitory receptor recognition of MCMV on CD8 T cells in genetically defined MHC I Dk disparate mice. We observed that Dk-licensed Ly49G2+ NK cells stabilized and then enhanced conventional dendritic cells (cDCs) recovery after infection. Furthermore, licensed NK support of cDC recovery was essential to enhance the tempo, magnitude, and effector activity of virus-specific CD8 T cells. Minimal cDC and CD8 T-cell number differences after low-dose MCMV in Dk disparate animals further implied that licensed NK recognition of MCMV imparted qualitative cDC changes to enhance CD8 T-cell priming.

Description: Abstract 3752 Background: Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment. Results: We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented. Conclusions: Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management. Disclosures: Brown: Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.