Abstract and Introduction
Abstract
Introduction: The increasing number of targeted therapies, together with a deeper understanding of cancer genetics and drug response, have prompted major healthcare centers to implement personalized treatment approaches relying on high-throughput tumor DNA sequencing. However, the optimal way to implement this transformative methodology is not yet clear. Current assays may miss important clinical information such as the mutation allelic fraction, the presence of sub-clones or chromosomal rearrangements, or the distinction between inherited variants and somatic mutations. Here, we present the evaluation of ultra-deep targeted sequencing (UDT-Seq) to generate and interpret the molecular profile of 38 breast cancer patients from two academic medical centers.
Methods: We sequenced 47 genes in matched germline and tumor DNA samples from 38 breast cancer patients. The selected genes, or the pathways they belong to, can be targeted by drugs or are important in familial cancer risk or drug metabolism.
Results: Relying on the added value of sequencing matched tumor and germline DNA and using a dedicated analysis, UDT-Seq has a high sensitivity to identify mutations in tumors with low malignant cell content. Applying UDT-Seq to matched tumor and germline specimens from the 38 patients resulted in a proposal for at least one targeted therapy for 22 patients, the identification of tumor sub-clones in 3 patients, the suggestion of potential adverse drug effects in 3 patients and a recommendation for genetic counseling for 2 patients.
Conclusion: Overall our study highlights the additional benefits of a sequencing strategy, which includes germline DNA and is optimized for heterogeneous tumor tissues.
Introduction
The use of highly effective targeted therapies in cancer frequently depends on the specific mutational profile of the tumor. As an increasing number of targeted therapies become available, determining the comprehensive genetic profile of a tumor is critical in understanding the response to targeted drugs for cancer treatment. Indeed, this genetic profile can help predict sensitivity or resistance to particular therapies and can therefore offer new, tailored treatment options to patients with late-stage or recurrent disease. In breast cancer, for example, trastuzumab has been used for Her2 amplified or overexpressing breast cancer. Notably, this strategy may suggest the use of a drug indicated for another anatomic cancer type, or the use of an investigational drug. Measuring the true clinical benefit of this tailored strategy is difficult, however, because targeted therapy frequently leads to drug resistance, the mechanisms of which are often not well understood. Nevertheless, this area of research is developing rapidly and some preliminary studies matching therapy to the tumor mutational profile across many clinical trials show an improved response rate.[1]
Traditionally, several types of molecular assays are available to identify somatic DNA mutations in tumors. Such assays analyze single positions, single exons, or whole genes using mass spectrometry,[2] allele-specific polymerase chain reaction (PCR)[3] or Sanger sequencing. These assays are, however, limited in scope – looking only at specific genes or mutations – and limited in sensitivity – usually dependent on the fraction of tumor cells contained in the tissue specimen. More recently, high-throughput sequencing of candidate genes has extended the breadth and sensitivity of this approach, overcoming some of these drawbacks.[4–7] Some major clinical centers are now starting to use more comprehensive molecular profiling in clinical care. However, these assays differ with regards to breadth (number of genes), depth (number of independent DNA molecules sampled) and design – selection of the genes or inclusion of a matched germline control. As a consequence, the clinical utility may vary. The Cancer Genome Atlas (TCGA),[8] a consortium focused on research and discovery, sequenced the entire exome of tumors but at limited coverage depth, rejecting specimens with less than 60% cellularity and preventing the reliable identification of subclonal mutations. More targeted commercial assays such as Foundation One (Foundation Medicine, Cambridge, MA) may generate increased coverage depth of a smaller set of genes but do not always report the mutant allelic fraction.[9] Such diagnostic services also omit the comparison with a matched germline control, which is essential to increase the analytical sensitivity and distinguish between inherited variants and somatic mutations.
Ultra-deep targeted sequencing (UDT-Seq)[5,10] of matched tumor–germline specimens has not yet been evaluated in a clinical setting. The sequencing of matched tumor–germline samples is crucial to distinguish somatic mutations from sequencing artifacts; it is also critical to establish with certainty that a variant identified in the tumor is somatic rather than inherited since filtering against polymorphism databases can eliminate real mutations.[11] In the absence of a matched germline DNA sequence, the misinterpretation of an inherited variant for a somatic mutation could potentially prevent a patient from getting appropriate genetic counseling. Additionally, inherited variation in metabolism genes such as DPYD or CYP2D6 has been associated with 5-fluorouracil toxicity and possibly tamoxifen efficacy,[12] respectively, and, although the variants are rare, a more systematic clinical screening would provide important benefits. The simultaneous sequencing of the germline DNA along with the tumor DNA therefore offers technical advantages to identify somatic mutations at low allelic fraction and increases the opportunity to identify actionable inherited variants.
Here, we evaluate a targeted sequencing assay for its use in a cancer clinical setting. Specifically, we performed UDT-Seq of 47 genes that are clinically actionable or important for patient care. We show that potentially important information is gained by sequencing at high depth, including identification of subclonal mutations. Additional information is also gained from the sequencing of matched germline DNA and from the inference of tumor DNA copy number alterations. We therefore demonstrate that in comparison with other high-throughput sequencing methods, UDT-Seq of matched tumor–germline DNA used in a clinical setting generates more potentially actionable findings for a greater number of patients.
Breast Cancer Res. 2013;15(6) © 2013 BioMed Central, Ltd.
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