Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20 degrees and 31 degrees tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.

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Background: Copy number (CN) studies of cancer hold great promise for the discovery of reliable biomarkers that can predict clinical outcomes such as prognosis and response to medication. One of the greatest challenges in the study of cancer CN (CCN) is that the vast majority of banked samples are formalin-fixed paraffin- embedded (FFPE). Due to DNA degradation, FFPE samples generally perform poorly with most CN technologies. However, MIP technology works well on FFPE samples, requiring only small stretches of intact genomic DNA (40-60 bp) in as little as a 75-ng input. We have applied this 330,000-plex platform on thousands of FFPE samples from various tumor tissue types, including breast, colon, ovary and brain, with more than 90% overall pass rate. This new platform allows an improved whole genomic coverage to an average of 10 kb-per-probe with a dense coverage of cancer-relevant coding regions. Experimental Design: MIP probes were synthesized, pooled and screened for their performance. A select set of 368,000 probes were chosen and used in the final chip design. The selection criteria were: (1) good quantitative performance, in terms of reproducibility with small variation in experiment metrics and in terms of the responsive dynamic range; (2) nonredundant whole genome coverage with minimum gaps (the maximum gap is limited to 100 kb) that includes additional dense coverage for relevant oncogenic regions. Our platform employs a measurement comparing neighboring markers across the entire genome: this median of absolute pairwise distribution, or MAPD, is a reliable metric for assessing tumor sample quality in the MIP-CCN assay. For array design, tag sequences were switched to genomic sequences with minimal impact on CN assessment. The best sequence of each interrogated SNP region was chosen based on empirical screening data. For the protocol, since each MIP probe hybridizes independently, the assay design should scale up for the larger 300K-plex probe panel. Indeed, the 330,000 probes worked well within the existing protocol with only minor modifications. Results: Using only 75 ng of input genomic DNA, we have obtained both good genotyping and CN data with the current 330K- probe platform. For cell-line DNA, CN quantitation performs with the same high quality as proven by 1X to 5X titration experiments. For FFPE samples, a dynamic range of up to 100 copies has been achieved. 0.6, a 2000-FFPE sample project has an overall pass rate £ Good concordance between normal and tumor sample pairs is also observed. Using an arbitrary cutoff of MAPD > 92%. In addition to CN information, data for hundreds of single-base somatic mutations are also generated. These markers are currently under validation. Conclusion: We have developed a powerful 330K MIP-CCN platform that works well on both fresh frozen/cell-line and archival FFPE samples. Currently we are applying the new platform to dozens of custom FFPE projects. References: 1. Wang Y et al. Performance of molecular inversion probes (MIP) in allele copy number determination. Genome Biol. 2007;8(11):R246. 2. Wang Y et al. High-quality copy number and genotype data from FFPE samples using molecular inversion probe (MIP) microarrays. BMC Med Genomics. 2009 Feb 19;2:8. 3. http://www.affymetrix.com/index.affx; search for “MIP” Citation Information: Clin Cancer Res 2010;16(14 Suppl):B14.

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The backbone torsion angle pair (φ, ψ) at each amino acid of a polypeptide is a descriptor of its conformation. One can use chemical shift and dipolar coupling data from solid-state NMR PISEMA experiments to directly calculate the torsion angles for the membrane-spanning portion of a protein.However, degeneracies inherent in the data give rise to multiple potential torsion angles between two adjacent peptide planes (a diplane). The molecular backbone structure can be determined by gluing together the consecutive diplanes, as in the PIPATH algorithm [25]. The multiplicities in torsion angles translate to multiplicities in diplane orientations. In this paper, we show that adjacent diplanes can be glued together to form a permissible structure only if they satisfy continuity conditions, described quantitatively here. These restrict the number of potential torsion angle pairs. We rewrite the torsion angle formulas from [22] so that they automatically satisfy the continuity conditions. The reformulated torsion angle formulas have been applied recently in the PIPATH algorithm [25] and will be helpful in other applications in which diplane gluing is used to construct a protein backbone model.

1434 Background: The prognostic significance of minimal residual detection (MRD) in both leukemia and lymphoma has been demonstrated in multiple cohorts. We will present a novel clinical assay, ClonoSIGHT, which leverages the advances in throughput and cost of DNA sequencing for T and B cell enumeration based on the deep sequencing of immunoglobulin and T-cell receptor rearrangements. This standardized clinical assay can be used for routine clinical monitoring of MRD in acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). The LymphoSIGHT platform for the universal amplification of immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up DNA samples has been presented previously. In this study, we demonstrate the technical performance of this technology as a clinical laboratory test. Methods: Primer sets for the amplification of variable (V), diversity (D), and joining (J) gene segments from genomic DNA for IgH@ have been demonstrated. These primer sets have been augmented by the development of universal primers capable of amplifying the full range of immune cell receptor rearrangements (D-J rearrangements for IgH@, and V-J rearrangements for IgK@, TCRB@,TCRG@, and TCRD@). Using these universal primer sets, we amplify rearrangements from genomic DNA in diagnostic and follow-up DNA samples from ALL and MCL patients. In order to make the platform compatible with routine clinical use, these amplicons are sequenced using a fast turnaround time platform (MiSeq from Illumina) to obtain multiple reads per sample enabling a 7 day time to result. Malignant clonotypes are automatically determined using diagnostic material (bone marrow or peripheral blood) and quantitated in follow up samples using standardized algorithms. In order to make these measurements clinically actionable, methods for determining the absolute number of malignant cells were developed and tested using artificially constructed samples. Sensitive methods for directly confirming the lack of sample to sample contamination were developed, which are critical to the integrity of a high sensitivity test. Results: The absolute quantitation of the IgH V-J assay has been demonstrated using dilution experiments from 12 ALL patients carrying 13 leukemic IgH clonotypes. The assay unequivocally detected leukemic signatures in all dilutions with expected concentration of at least one leukemic cell in 1 million leukocytes (a sensitivity of 10−6) (Figure 1). High precision was illustrated by low random error (4.1% to 7.6% average relative standard deviation at clonotype frequencies at or above 3×10−5). For each clonotype, the assay showed high r2 values with a range of 0.977 and 0.996 (mean 0.988, median 0.991) between each of the expected and measured clonotype frequencies. The slopes ranged from 0.878 to 1.14 (mean 1.00, median 0.977), illustrating the quantitative nature of the assay over at least 3 orders of magnitude. In an analysis of all clonotypes together, we measured an r2 value of 0.94 and a slope of 1.01 between the expected and measured clonotype frequencies. The ability to identify malignant clonotypes in multiple receptors has been demonstrated in ALL and MCL clinical samples. The ability to detect multiple clonotypes within a calibrating receptor has also been demonstrated in these clinical samples; data will be presented. We will also describe the laboratory workflow and quality system that has been developed, as well as standardized algorithmic approaches to measure contamination in every sample and sequencing run, to ensure high fidelity of the data. Conclusions: In order for a novel MRD assay to be clinically useful, it must exhibit superior performance compared with existing platforms and be capable of reproducibly measuring absolute MRD levels across multiple receptors in a rapid turnaround fashion with high data integrity and without any cross contamination of samples. The ClonoSIGHT assay has demonstrated these qualities and can thus be considered for clinical use. Regulatory infrastructure, laboratory process controls, quality systems, and standardized documentation associated with CLIA-approved laboratories have been developed. Validation results will be presented. Disclosures: Willis: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Asbury:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Carlton:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Klinger:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Pothuraju:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Weng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.