David B. Lieberman

Detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next generation sequencing of cell-free circulating tumor DNA.

Purpose: The expanding number of targeted therapeutics for non–small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS. Experimental Design: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible. Results: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible. Conclusions: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772–82. ©2016 AACR.

Using “residual” FNA rinse and body fluid specimens for next‐generation sequencing: An institutional experience

Tissue specimens are typically considered optimal for molecular testing; however, in the current era of personalized medicine, cytopathology specimens are increasingly recognized as potential sources for molecular testing. This is often accomplished by using cell block specimens and/or fine‐needle aspiration (FNA) smear preparations. In this study, the authors investigated the feasibility, performance, and quality of “residual” FNA rinse and body effusion fluids used for next‐generation sequencing (NGS).

A novel approach for next-generation sequencing of circulating tumor cells.

Next‐generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low‐input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching.

Clinical Utility of Next-Generation Sequencing for Oncogenic Mutations in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation.

To determine the association of somatic mutations in acute myeloid leukemia (AML) with risk of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT), we retrospectively studied pre-transplantation genetic profiles obtained from next-generation sequencing of 26 genes in 112 adult patients with AML who underwent alloHSCT. Univariable and multivariable regression analyses were used to assess the association between the presence of a pathogenic mutation and risk of relapse after alloHSCT. Eighty-six percent (96 of 112) of patients had at least 1 pathogenic mutation. Mutations in TP53, WT1, and FLT3-internal tandem duplication (ITD) were associated with an increased risk of relapse after alloHSCT (adjusted hazard ratio [aHR], 2.90; P = .009; aHR, 2.51; P= .02; and aHR, 1.83; P = .07, respectively). DNMT3A mutation in the absence of FLT3-ITD and NPM1 mutations was associated with a lower relapse risk (aHR, .22; P = .04). Comparison of pre-alloHSCT and post-alloHSCT genetic profiles showed clonal evolution in 6 of 6 patients, including acquisition of actionable mutations in 4 patients. In summary, genetic profiling is useful for assessing relapse risk in patients with AML undergoing alloHSCT and may identify patients in need of strategies to reduce this risk. Clonal evolution is present at post-alloHSCT relapse and repeat genetic profiling may uncover acquired actionable mutations.

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

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 (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). 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 FLT3 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.

NPM1 Mutation is Associated with Leukemia Cutis in Acute Myeloid Leukemia with Monocytic Features

Leukemia cutis (LC), the infiltration of the epidermis, dermis, or subcutis with leukemia cells, complicates 5–10% of cases of acute myeloid leukemia (AML) in adults and is considered a marker of poor prognosis.[1][1]–[3][2] While the association between AML with monocytic features and LC has

Abstract 4916: Development of a NGS-based method for EGFRvIII detection: sequence analysis of the junction

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary tumor in humans. One of the most common mutations in GBMs is an interstitial deletion in the epidermal growth factor receptor (EGFR), EGFRvIII, which occurs at a frequency of ∼30%. EGFR is a transmembrane tyrosine kinase receptor and the EGFRvIII mutant is characterized by a deletion of 267 amino acids in the extracellular domain leading to ligand independent constitutive activation. The deletion of exons 2-7 leads to an in-frame deletion in EGFR with a novel glycine residue at the junction. The amino acid at the junction of exons 1 and 2 is a valine, making the novel transcript an attractive target for immunotherapy. A custom next generation sequencing (NGS) based assay and bioinformatic pipeline have been developed in our laboratory to detect EGFRvIII from RNA extracted from formalin fixed paraffin embedded tissue. The targets include the exon 1-2 boundary (wild type), the exon 1-8 boundary (EGFRvIII), amplification of various sized RNA fragments to determine RNA degradation and bioavailability, and expression levels of three housekeeping genes. Following cDNA synthesis multiplex PCR of all targets are captured simultaneously for the sequencing library with NGS performed on the Illumina MiSeq. The output from the bioinformatics pipeline includes the sequence and number of reads from the wild-type and mutant, ratio of EGFRvIII reads with respect to total EGFR sequenced, expression of three housekeeping genes and relative amount of bioavailable EGFR RNA. This assay was validated through comparison of NGS sequence results with an established qRT-PCR to detect normal and mutant EGFR. Negative controls from normal brain (temporal lobe excisions from epilepsy patients) and adipose tissue (a tissue with high expression of EGFR) were used to determine whether low-level exon 1-8 fusions from mis-splicing were detectable in normal tissue (Figure 1). Twenty five GBM specimens were sequenced, with 8/25 positive for EGFRvIII (Figure 2), and confirmed by RT-PCR. In addition to detection of the EGFRvIII mutant, relative expression of EGFR is detected in this assay, and when taken together with EGFR amplifications detected by routine NGS panels, we can determine whether the EGFRvIII is present on the amplified or unamplified allele and whether additional mutations are detectable. Detection of EGFRvIII utilizing NGS improves the precision of mutant detection to better serve CART-EGFRvIII clinical trial to ensure the target is present. The NGS assay provides the EGFRvIII/wild-type ratio, relative expression levels for EGFR and EGFRvIII and evaluation of RNA degradation in a single assay. Figure 1A. Baseline in normal samples. EGFRvIII ratio in 18 “normal” brain and 11 adipose tissue samples, plotted without (top panel) and with (bottom panel) a EGFRvIII positive sample. Figure 2. EGFRvIII ratio in 25 GBM samples. Cutoff for EGFRvIII positive is EGFRvIII ratio of 0.3 (30%). Citation Format: Jianhua Zhao, Shrey Sukhadia, Alan Fox, David Lieberman, Barnett Li, Robert D. Daber, Matthew C. Hiemenz, David B. Roth, Maria Martinez-Large, Arati Desai, Donald M. O9Rourke, Marcela V. Maus, Jennifer JD Morrissette. Development of a NGS-based method for EGFRvIII detection: sequence analysis of the junction. [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 4916. doi:10.1158/1538-7445.AM2015-4916

Feasibility of monitoring advanced melanoma patients using cell-free DNA from plasma

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell‐free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra‐deep sequencing for a 61‐gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next‐generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%.

Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non–Small Cell Lung Cancer

Importance The clinical implications of adding plasma-based circulating tumor DNA next-generation sequencing (NGS) to tissue NGS for targetable mutation detection in non–small cell lung cancer (NSCLC) have not been formally assessed. Objective To determine whether plasma NGS testing was associated with improved mutation detection and enhanced delivery of personalized therapy in a real-world clinical setting. Design, Setting, and Participants This prospective cohort study enrolled 323 patients with metastatic NSCLC who had plasma testing ordered as part of routine clinical management. Plasma NGS was performed using a 73-gene commercial platform. Patients were enrolled at the Hospital of the University of Pennsylvania from April 1, 2016, through January 2, 2018. The database was locked for follow-up and analyses on January 2, 2018, with a median follow-up of 7 months (range, 1-21 months). Main Outcomes and Measures The number of patients with targetable alterations detected with plasma and tissue NGS; the association between the allele fractions (AFs) of mutations detected in tissue and plasma; and the association of response rate with the plasma AF of the targeted mutations. Results Among the 323 patients with NSCLC (60.1% female; median age, 65 years [range, 33-93 years]), therapeutically targetable mutations were detected in EGFR, ALK, MET, BRCA1, ROS1, RET, ERBB2, or BRAF for 113 (35.0%) overall. Ninety-four patients (29.1%) had plasma testing only at the discretion of the treating physician or patient preference. Among the 94 patients with plasma testing alone, 31 (33.0%) had a therapeutically targetable mutation detected, thus obviating the need for an invasive biopsy. Among the remaining 229 patients who had concurrent plasma and tissue NGS or were unable to have tissue NGS, a therapeutically targetable mutation was detected in tissue alone for 47 patients (20.5%), whereas the addition of plasma testing increased this number to 82 (35.8%). Thirty-six of 42 patients (85.7%) who received a targeted therapy based on the plasma result achieved a complete or a partial response or stable disease. The plasma-based targeted mutation AF had no correlation with depth of Response Evaluation Criteria in Solid Tumors response (r = −0.121; P = .45). Conclusions and Relevance Integration of plasma NGS testing into the routine management of stage IV NSCLC demonstrates a marked increase of the detection of therapeutically targetable mutations and improved delivery of molecularly guided therapy.

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.