Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing

<div><p>Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (<i>EGFR</i>, <i>KRAS</i>, <i>BRAF</i>, <i>NPM1</i>, <i>FLT3</i>, and <i>JAK2</i>). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two <i>FLT3</i> internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.</p></div

Next-generation sequencing data analysis pipeline.

<p>Data analysis occurs in three sequential stages, pre-processing of NGS reads, variant calling, and variant annotation. Of note, large indels are detected by an examination of reads that failed to map to target regions of the reference genome and are recovered from a pool of rejected reads (“Trash”). SNVs, single nucleotide variants. CNVs, copy number variation.</p

Two Specimens with Low-Allele Frequency <i>FLT3</i> Internal Tandem Duplications.

<p>In one specimen (A and B), a 24 bp internal tandem duplication (ITD) was seen in 7 out of 529 reads for an allele frequency of 1.3%. (A) Four of the reads containing insertions (purple bars) are shown using the Integrative Genomics Viewer. This specimen additionally harbored a <i>FLT3</i> D839G mutation in 45% of reads (B). A second specimen (C) harbored a 33 bp <i>FLT3</i> ITD in 12 out of 739 reads, for an allele frequency of 1.6%. Nine of the reads carrying an ITD are pictured.</p

Concordance Analysis of Solid Tumor Specimens.

<p>The specimens shown were submitted for both NGS and targeted tests for <i>EGFR</i> (A), <i>KRAS</i> (B), and <i>BRAF</i> (C) mutations. Note that all mutations seen by targeted testing were also found by NGS when specimens with inadequate DNA quantity and/or quality are excluded.</p

Tumor Percentage of Specimens Analyzed by Targeted <i>EGFR</i> Tests.

<p>Cases were placed into 5 bins (<5%, 5–10%, 11–25%, 26–50%, >50% tumor cells in specimen).</p

Proposed Workflow for NGS and Single-Gene Assays.

<p>Three main decision points are highlighted. Specimens requiring an urgent turnaround time are routed directly for single-gene testing (possibly followed by NGS). Additionally, single-gene testing is performed on samples with less than 10% tumor or DNA inadequate for NGS (i.e., degraded or low quantity). In samples not meeting any of the above criteria, NGS is performed instead of single-gene testing. NGS results do not require confirmation by single-gene testing.</p

Characteristics of Specimens by Tumor Site.

<p>Characteristics of Specimens by Tumor Site.</p

Concordance Analysis of Hematologic Malignancy Specimens.

<p>The specimens shown were submitted for both NGS and targeted tests for <i>FLT3</i> (A), <i>NPM1</i> (B), and <i>JAK2</i> (C) mutations. Note that all samples were adequate for testing by both single-gene assays and NGS.</p

Tumor Percentage of Solid Tumor Specimens.

<p>Specimens were analyzed by next-generation sequencing (NGS) within the study period of March 1, 2013 and March 1, 2014.</p

Overall survival in IGP model subgroups.

<p>A. Overall survival by cytogenetics and mutational profiles. Among patients with intermediate cytogenetics, three-year overall survival was 59% for those with favorable mutational profiles (A), 33% for those with intermediate mutational profiles (B), 51% for those who were <i>FLT3-</i>ITD negative with high-risk mutations (C) and 11% for those who were <i>FLT3-</i>ITD positive with high-risk mutations (D). Three-year overall survival was 77% among patients with favorable cytogenetics (favorable) and 21% among patients with unfavorable cytogenetics (unfavorable). B. Overall survival among patients with favorable mutational profiles. The overall survival curve for patients with intermediate cytogenetics and mutant <i>NPM1</i> plus mutant <i>IDH1</i> or <i>IDH2</i> was similar to the survival curve for patients with favorable cytogenetics (adjusted p = 0.697) and different from the survival curve for patients in the intermediate IGP risk group (adjusted p = 0.028). C. Overall survival among patients with <i>FLT3-</i>ITD negative AML and high-risk mutations. The overall survival curve for patients with <i>FLT3</i>-ITD negative (<i>FLT3-</i>ITD-) AML and co-occurring high-risk mutations (<i>TET2</i>, <i>ASXL1</i> and/or <i>PHF6</i>) was not significantly different from the survival curves for patients with unfavorable cytogenetics or patients with intermediate IGP risk (adjusted p = 0.111 and p = 0.919, respectively). D. Overall survival among patients with <i>FLT3</i>-ITD positive AML and high-risk mutations. The overall survival curve for patients with <i>FLT3</i>-ITD positive (<i>FLT3</i>-ITD+) AML and co-occurring high-risk mutations (trisomy 8, <i>TET2</i> and/or <i>DNMT3A</i>) was similar to the survival curve for patients with unfavorable cytogenetics (adjusted p = 0.793) and different from the survival curve for patients in the intermediate IGP risk group (adjusted p = 0.022).</p