Danielle Perrin

MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.

A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.

Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (∼1.5–5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing.

Abstract 3152: CanSeq: prospective clinical whole-exome sequencing of FFPE tumor samples.

INTRODUCTION: Knowledge of tumor genomic alterations that may predict therapeutic responses represents a growing clinical need. Although there has been an increase in the use of hotspot genotyping and targeted sequencing panels of small numbers of genes, the use of clinical whole exome sequencing (WES) remains underdeveloped. Prospective WES in cancer patients presents several logistical and scientific challenges, including (i) generating robust sequencing data from small amounts of FFPE tumor tissue, (ii) establishing a clinically relevant turnaround time, (iii) achieving clinical interpretation of genomic alterations, and (iv) communicating results to the clinical team and patients. Here, we describe CanSeq, a clinical WES platform to detect genomic alterations in FFPE tumor samples and identify clinically actionable and biologically meaningful alterations in order to aid clinical decision-making and inform future research. METHODS: We performed prospective WES on tumor and germline DNA from patients with advanced refractory cancer. Sequencing was performed at the Broad Institute using the Illumina HiSeq, starting with 100 ng of DNA or less from FFPE tumor tissue and matched normal blood. Data were analyzed using a novel algorithm to highlight clinically actionable mutations, indels, and copy number alterations. These were annotated and assigned levels of evidence, and an interactive web-based report was generated for review by clinicians. RESULTS: The average turnaround time from sample receipt to data delivery was 16 days. Each tumor sample was sequenced at 90X or greater depth of coverage with more than 80% of exons with at least 30X coverage. Analysis of the first 15 patients revealed at least one plausibly actionable somatic mutation in 14 samples. These alterations include “standard of care” alterations (BRAF, EGFR), entry criteria for clinical trials (PIK3CA, KRAS, PTEN), potentially actionable alterations based on more limited evidence (STK11, ATM, CRKL, CTNNB1, PDGFRA, CDK4, CDKN2A, SMARCB1, TP53), and alterations that were theoretically targetable (JAK3, SYK). Additional biologically relevant somatic alterations and notable germline alterations were also identified. For several patients, actionable alterations were confirmed in a CLIA lab and impacted clinical decision-making, including enrollment in clinical trials. CONCLUSION: We have developed a prospective clinical WES platform to robustly detect genomic alterations in archival FFPE tumor samples (using Citation Format: Nikhil Wagle, Eliezer Van Allen, Danielle Perrin, Dennis Friedrich, Sheila Fisher, Gregory Kryukov, Lauren Ambrogio, Daniel Auclair, Stacy Gray, Steven Joffe, Pasi Janne, Judy Garber, Laura Macconaill, Neal Lindeman, Barrett Rollins, Phillip Kantoff, Gad Getz, Stacey Gabriel, Levi A. Garraway. CanSeq: prospective clinical whole-exome sequencing of FFPE tumor samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3152. doi:10.1158/1538-7445.AM2013-3152