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Purpose. The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. Methods. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls.Results. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency .5% and demonstrated highlevel molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "falsepositive" calls in clinically druggable oncogenes such as PIK3CA. Conclusion. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making. The Oncologist 2014;19:336-343 Implications for Practice: Next-generation sequencing technologies permit deep sequencing of hundreds of cancer genes concurrently, offering an unprecedented opportunity to identify the clinically relevant mutations in personalized cancer care.The Ion AmpliSeq Cancer Panel provides a rapid and cost-effective sequencing workflow for single-tube preparation of amplicon libraries from genomic "hotspot" regions that are frequently mutated in human cancer genes.This study evaluates the advantages and disadvantages of the AmpliSeq platform for clinical applications, and describes how this new knowledge can be used to ensure the accuracy of mutation detection for precision oncology.

Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the PTEN tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (AR) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an in silico down-sampling approach, we determined that DNA copy number and genomic instability can be detected with as few as 350K sequencing reads. The data shown here demonstrate the feasibility of detecting genomic instabilities at the single cell level using the Epic Sciences CTC Platform. Understanding CTC heterogeneity has great potential for patient stratification prior to treatment with targeted therapies and for monitoring disease evolution during treatment.

Multiple myeloma (MM) remains an incurable disease despite recent therapeutic improvements. The ability to detect and characterize MM circulating tumour cells (CTCs) in peripheral blood provides an alternative to replace or augment invasive bone marrow (BM) biopsies with a simple blood draw, providing real-time, clinically relevant information leading to improved disease management and therapy selection. Here we have developed and qualified an enrichment-free, cell-based immunofluorescence MM CTC assay that utilizes an automated digital pathology algorithm to distinguish MM CTCs from white blood cells (WBCs) on the basis of CD138 and CD45 expression levels, as well as a number of morphological parameters. These MM CTCs were further characterized for expression of phospho-ribosomal protein S6 (pS6) as a readout for PI3K/AKT pathway activation. Clinical feasibility of the assay was established by testing blood samples from a small cohort of patients, where we detected populations of both CD138pos and CD138neg MM CTCs. In this study, we developed an immunofluorescent cell-based assay to detect and characterize CTCs in MM.

5508 Background: ctDNA can inform on prognosis, treatment response and survival. We evaluated ctDNA in serial plasma samples from patients enrolled in A.MARTIN (NCT01485861), a randomized phase II study of abiraterone with or without ipatasertib in patients with mCRPC. Methods: Blood was collected in cell-free DNA Streck tubes from 216 patients at 3 time points; baseline, C3D1 and end of treatment. Cell-free DNA (cfDNA) was extracted from plasma using a Circulating DNA Kit (Qiagen) on a QIASymphony machine (Qiagen). 25ng of extracted cfDNA was used in library preparation, constructed with a custom designed, 58 gene, QIAseq Targeted DNA panel (Qiagen) enriched for PI3K/AR pathway genes. Samples were sequenced to mean depth of 3394x on a NextSeq500 machine. Unless otherwise noted, all analyses combine patients across the 3 study arms, and reported p-values are unadjusted. Results: Baseline (BL) ctDNA positivity correlated with radiological progression-free survival (rPFS; HR: 1.8 [95% CI 1.3-2.6], p < 0.01); this association with rPFS was maintained in a multivariate cox model with > 5 baseline clinical variables (HR: 1.6 [95% CI 1.1-2.4]; p = 0.011). Patients with a C3D1 reduction in ctDNA had superior rPFS compared to patients with a C3D1 increase in ctDNA (HR: 2 [95% CI 1.3-3.2], p < 0.01). The rate of ctDNA clearance at C3D1 was higher in the Ipatasertib 400mg arm compared to placebo (56.3% versus 24.4%, p < 0.01). We find that changes in ctDNA associated with best confirmed overall response (p = 0.024); CR patients had the greatest reduction in ctDNA (mean of -23.4%), followed by PR (-16.3%), then SD (-4.1%), and lastly PD patients (-1.3%). Changes in ctDNA levels correlated with SLD changes (rs = 0.289, p = 0.05), and also PSA changes (rs = 0.33, p < 0.01). Changes in ctDNA were associated with rPFS in a multivariate cox analysis that included PSA change (p < 0.01), as well as in a separate multivariate analysis that included SLD change (p < 0.01). Lastly, we explored CNVs and observed emerging resistance mutations in progression samples, including alterations in TP53, AR, FOXA, PTEN, and PI3K/AKT pathway genes. Conclusions: ctDNA analyses may help (i) identify poorer prognosis disease at baseline, (ii) inform on treatment response (CR/PR/SD/PD) and radiological progression free survival (rPFS) in on-treatment (C3D1) samples, and (iii) can elucidate emerging resistance mechanisms at disease progression. Clinical trial information: NCT01485861 .