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Pools of cell lines carrying a variety of known mutations are used to validate the performance of a cancer diagnostic
test based on next-generation sequencing.
As next-generation sequencing (NGS) of tumor
cells becomes more sophisticated, it is likely to
inform all aspects of cancer management, from
diagnostic testing to treatment and drug discovery. In this issue, Frampton et al.1 describe a
comprehensive NGS assay applicable to clinical
samples that identifies single-base substitutions,
copy-number variations and focal amplifications in 287 cancer-related genes, fusion events
involving 19 frequently rearranged genes and
3,549 single-nucleotide polymorphisms in
other locations throughout the genomeall
within a single sequencing run. Validating the
analytic performance of such a test is challenging because it assays so many nucleotide
positions in the genome (~1.5 Mb in total).
The authors therefore use 53 cell lines to create
reference materials for assessing the sensitivity and specificity of variant detection. Finally,
they apply their test to >2,000 clinical cases.
The overall approach of the studyincluding
assay design, creation of reference materials and
validationserves as a model for the development of future clinical NGS tests.
In current clinical practice, mutation analysis of cancer samples is performed to establish diagnoses (e.g., specific translocations
Jason Y. Park is at the Department of Pathology,
University of Texas Southwestern Medical
Center and Childrens Medical Center, Dallas,
Texas, USA, and the Eugene McDermott
Center for Human Growth and Development,
University of Texas Southwestern Medical
Center, Dallas, Texas, USA; Larry J. Kricka is
at the Department of Pathology and Laboratory
Medicine, University of Pennsylvania
Medical Center, Philadelphia, Pennsylvania,
USA; and Paolo Fortina is at the Cancer
Genomics Laboratory, Kimmel Cancer Center,
Department of Cancer Biology, Thomas
Jefferson University, Jefferson Medical College,
Philadelphia, Pennsylvania, USA.
e-mail: jaspar@childrens.com
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n e w s and v i e w s
Tumor sample
in paraffin block
Pathology review
DNA extraction
Library preparation
Paired-end sequencing
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Variant calling
Base substitutions
Variant type
Reference material
used for validation
Copy-number variations
7 discrete tumor cell lines
42 pools from combinations of tumor
cell lines and normal cell lines
Each tumor cell line tested from 0 to 80%
dilution with normal cell lines
Figure 1 Clinical sequencing test protocol and validation. Patient tumor samples are received as
formalin-fixed, paraffin-embedded blocks, which are from diagnostic surgical pathology. These types
of samples are heterogeneous mixtures of neoplastic and normal cells. Frampton et al.1 design DNA
probes for solution capture of ~1.5 Mb of genomic regions known to be related to tumorigenesis. Variants
are called from the sequencing data using bioinformatic algorithms. The analytical performance of the
assay is demonstrated by manufacturing and testing custom reference materials derived from pooled
cell lines. The pooled cell lines contain genomic DNA alterations that represent a wide spectrum of base
substitutions, insertions and deletions, and copy-number variations. Reference materials for insertions
and deletions and copy-number variations share two cell lines. MAF, mutant allele frequency.
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n e w s and v i e w s
1. Frampton, G.M. et al. Nat. Biotechnol. 31, 10231031
(2013).
2. Conti, R.M. et al. J. Clin. Oncol. 31, 11341139 (2013).
3. Gargis, A.S. et al. Nat. Biotechnol. 30, 10331036 (2012).
4. Rehm, H.L. et al. Genet. Med. 15, 733747 (2013).
5. CAP Laboratory Accreditation Program. Molecular
Pathology Checklist http://www.cap.org/apps/docs/
laboratory_accreditation/checklists/new/molecular_
pathology_checklist.pdf (College of American
Pathologists, 2013).
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