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Circulating-free DNA (cfDNA) holds great potential for non-invasive somatic mutation detection in cancer patients as a so-called “liquid biopsy”. A liquid biopsy is especially valuable when tumor tissue is not available and for longitudinal monitoring of tumor burden or emerging resistance. In such cases, tumor-specific mutations are not known a priori and in order to detect tumor-derived somatic mutations it is best to interrogate a broad panel of cancer genes. The primary obstacle to sequencing cfDNA with broad mutation panels is achieving the necessary limit of detection to identify small amounts of tumor-derived cfDNA relative to the predominant wild-type cfDNA in the plasma of cancer patients. Tumor-derived cfDNA levels correlate with tumor stage, also making it an important consideration when investigating the sensitivity of this approach. We demonstrate the detection of somatic variants in several cancer genes in the plasma of early and late-stage colorectal cancer (CRC) patients by deep sequencing (> 8,000X) the cfDNA and matched normal DNA of N=33 CRC patients using the AmpliSeq cancer panel. The AmpliSeq panel requires less than 10ng of input DNA and amplifies hotspot loci of 50 known cancer genes for next-generation sequencing (NGS). We observe at least one somatic mutation with greater than 0.1% variant allele frequency in the cfDNA of all 33 CRC patients. We also show the sensitivity of detecting tumor-derived somatic mutations in cfDNA by exome sequencing of the primary tumor tissue. We find higher sensitivity for tumor mutations in cfDNA of late-stage patients compared to early-stage patients. Overall, we demonstrate the feasibility of using NGS on a small sized cancer panel for identifying somatic mutations, including those with low variant allele frequency, in cfDNA of early and late-stage CRC patients. Although a cfDNA approach holds promise as a tool to aid in pre-clinical and clinical research, more work is needed to understand its performance under different assay and disease conditions. Citation Format: Steven M. Bray, Philip J. Ebert, John N. Calley, Richard E. Higgs, Isabella H. Wulur, Swee Seong Wong, Candice L. Horn, Ricardo Martinez, Christoph Reinhard. Concordance of somatic mutations found in primary tumors and plasma circulating-free DNA from early and late-stage colorectal cancer patients. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 29.

Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. [Mol Cancer Ther 2008;7(9):2955–66]

Thrombin cleavages of selective proteinase-activated receptors (PAR) as well as PAR-activating peptide ligands can initiate the phosphoinositide 3-kinase (PI3K) signaling cascade in platelets. Downstream to this event, fibrinogen receptors on platelets undergo conformational changes that enhance fibrinogen binding. In our study, we used this phenomenon as a surrogate biomarker for assessing effects on PI3K activity. Our method, using flow cytometric measurement of fluorescent ligand and antibody binding, uncovered a 16- to 45-fold signal window after PAR-induced platelet activation. Pretreatment (in vitro) with the PI3K inhibitors wortmannin and LY294002 resulted in concentration-dependent inhibition at predicted potencies. In addition, platelets taken from mice treated with wortmannin were blocked from PAR-induced ex vivo activation concomitantly with a decrease in phosphorylation of AKT from excised tumor xenografts. This surrogate biomarker assay was successfully tested (in vitro) on blood specimens received from volunteer cancer patients. Our results indicate that measurement of platelet activation could serve as an effective drug activity biomarker during clinical evaluation of putative PI3K inhibitors. [Mol Cancer Ther 2007;6(9):2600–7]

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Introduction: Plasma cell-free circulating tumor DNA (ctDNA) has the potential to serve as a noninvasive surrogate to determine tumor genotype and monitor treatment response. Here we report the analytical validation of the Ion AmpliSeq v2 Cancer Panel (ACP) in plasma ctDNA samples. Methods: Mutations were assessed using the ACP, designed to survey 2800 mutations in 50 cancer-related genes. TruQ reference standards were used to determine LOD. Healthy donor and cancer patient plasma samples were used for other studies. Eight libraries were barcoded and sequenced with PGM sequencing 200 kit v2 on a 318 v2 chip. The sequencing data were analyzed with a combination of Torrent Suite 3.4.2, VarScan, our proprietary analysis pipeline and IGV. The background noise level of hotspot nt positions was calculated from multiple runs of known wild type DNA. In order to increase the sensitivity of the assay to detect low-level mutation, a custom program was developed to detect variants clearly different from background noise (95% confidence) and not reported by the two variant callers. PCR methods were used for concordance testing. Results: The performance characteristics of the AmpliSeq v2 Cancer Panel were demonstrated in 1) Minimum DNA input and LOD: With 10 ng DNA input, 43/43 and 42/43 expected mutations were detected at 2.5% or 1%, respectively. With 1 ng DNA input, 42/43 and 40/43 expected mutations were detected at 2.5% and 1%, respectively. 2) Coverage: The mean library coverage for 40 plasma libraries was 2912x with only 5 non-critical regions below 250x coverage. 3) Baseline error rate: 95% of 2290 hot-spots for SBS showed Conclusions: The ACP offers a useful tool for comprehensive somatic mutation profiling in ctDNA. The limit of detection is 1-2% mutation frequency for most mutations with slight variation across different nucleotide positions. The low concentration of ctDNA obtained from plasma samples can present a challenge. Less than 10 ng DNA input may result in allele drop out for low level mutations due to the inefficient amplification of multiplex PCR compared with PCR methods using a short length single amplicon. Higher DNA input to rescue allele drop-out is under evaluation. Citation Format: WeiHua Liu, Zhenyu Yan, Candice L. Horn, Fabio Nunes, Steven M. Bray, Philip J. Ebert, Peng Fang, Jennifer Biroschak, Cindy Spittle, Chad Galderisi, Jin Li. Liquid biopsy using the ion AmpliSeq v2 cancer panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2015-4925

Introduction: Somatic mutations are routinely identified using NGS cancer panels but these panels lack genome-wide coverage for copy number (CN) and LOH analysis. To investigate mutation, CN, and LOH in late-stage breast cancer we tested >50 samples using the Oncoscan molecular inversion probe (MIP) array and evaluated its reproducibility and performance compared to NGS platforms. Methods: 52 breast cancer samples (stage IIIA - IV) were analyzed using MIP array (Oncoscan, Affymetrix). Four samples were tested in technical triplicates to determine assay reproducibility. In addition, 28 samples were sequenced by amplicon-based NGS and five of these samples were also tested using a capture-based NGS platform for mutation, CN, and LOH comparison. Results: MIP array provided highly reproducible results for CN and LOH, with >98% of calls showing CN range in the technical triplicates of < 0.5 copy for 891 cancer genes analyzed. Variability in CN seems to be proportional to absolute copy number at the tested locus, with CN range in technical triplicates of >2 copies seen in only two cases, once for ERBB2 (CN range 32 - 35 copies) and once for FLOT2 (CN range 22 - 25 copies). Gene level results were then categorized in five groups: homozygous deletion, single copy loss, diploid, low grade amplification (≤6 copies), or high grade amplification (>6 copies). Using these predetermined cut points, we saw >99% concordance rate among the technical replicates in the MIP array. We found a 93% concordance rate between MIP array and CN/LOH calls by capture-based NGS. Discordant calls between NGS and MIP array were either LOH calls or single copy number change (diploid vs. single copy loss or gain). MIP array mutation analysis of 28 samples showed good sensitivity, correctly detecting the 17 PIK3CA mutations and one TP53 mutation identified by NGS in this cohort. There were seven false positive calls by MIP array, five of them occurring in two genotypes (2x NRAS G12S/C, and 3x EGFR L858R). The other two false positives occurred in PIK3CA, with one false positive (H1047L) occurring in association with a high-grade PIK3CA amplification (7 copies). Increasing CN at the mutation locus was associated with a higher mutation score provided by MIP array (p Conclusions: MIP array platform provides a great alternative for assessing CN and LOH in FFPE samples at lower cost and using less input DNA than NGS (80ng vs. 250ng). There was good correlation between CN and LOH results from MIP array and capture-based NGS, with discordant results limited to small CN differences or LOH calls. Mutation analysis by MIP array showed no false negatives when compared to NGS, while false positives seem to occur either due to probe-specific issues or in association with amplifications at the genotyping locus. Citation Format: Candice L. Horn, Fabio Nunes, John Calley, Steven Bray, Isabella Wulur, Mark Farmen, Robert Gallavan, Iris Halfpenny, Paul Medlow, Keith McGreeghan-Crosby, Gera Jellema. Copy number and loss of heterozygosity (LOH) analysis in 52 breast cancer FFPE samples using molecular Inversion probe array: detailed analysis of reproducibility and performance compared to NGS platforms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-048. doi:10.1158/1538-7445.AM2015-LB-048

Anaplastic lymphoma kinase (ALK, Chr 2) is an orphan transmembrane receptor tyrosine kinase that is activated in non-small cell lung cancer (NSCLC), neuroblastoma, and anaplastic large cell lymphomas (ALCL). Genetic activation of ALK can occur as a result of gene amplification, kinase-domain point mutations, or by fusion of the kinase domain to structural genes such as echinoderm microtubule associated protein-like 4 (EML4). Ultimately this leads to further activation of downstream effector proteins-including STAT3, AKT or ERK-enhanced cell proliferation, and disease. A study of 602 cancer cell lines found that neuroblastoma, NSCLC and ALCL cells harboring ALK translocations and amplifications were particularly responsive to treatment with ALK inhibitor therapy (McDermott et al, 2008). Recently, somatic and germline missense mutations were also identified in the ALK kinase domain and linked to 8.4% of hereditary and spontaneous pediatric neuroblastomas (Mosse et al., 2008). The ALK mutations were found to cause an increase in ALK expression that when knocked down resulted in an inhibition of cancer cell growth (Mosse et al., 2008). These results suggest that ALK point mutations have similar functional consequences in neuroblastoma as ALK structural variations and could therefore confer cancer cell sensitivity to ALK inhibitor therapy. Although few studies have investigated this to date, the ALK point mutations may result in similar functional consequences for other cancer cell types. If so, the ALK mutations may be ideal genetic biomarkers to identify patients who would be clinically responsive to ALK inhibitor therapy. Therefore, we sought to better understand a subset of these ALK somatic mutations, their frequency in other cancer types, and potential as clinical biomarkers for cancer drug response. We designed and developed custom allelic discrimination assays to genotype three ALK activating mutations; M1166R, F1174L and R1275Q in DNA extracted from 23 cancer cell lines and 47 tumor samples. Although no neuroblastoma samples were included, these 70 samples represented a dozen different cancer types and tissues, including prostate, breast, ovary, lung, liver and pancreas. We also generated synthetic oligonucleotide allele controls to establish the necessary heterozygous and homozygous genotype clusters for automated genotype calling. Our data showed that the ALK mutations were monomorphic wildtype in all 70 cancer samples tested. These results suggest that the 12.4% frequency of the ALK mutations observed in neuroblastoma may be very specific and play a particular role in that disease. More studies are required to elucidate if these ALK mutations may provide tools for additional cancer patient stratification, including predicted disease progression and response beyond the previously identified ALK translocations and amplifications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2730.