Association for Molecular Pathology Annual Meeting 2017; Salt Lake City, UT; November 16-18, 2017 | ASC

Learn about the latest ONCOREF™ Precision Molecular Reference Standards at the 2017 AMP Annual Meeting

AccuRef Diagnostics, a division of Applied StemCell, will be at the AMP annual meeting to be held in Salt Lake City, UT from November 16-18, 2017. Please visit our booth (#904) and also our poster presentation to learn about the latest in precision molecular reference standards available for molecular diagnostics.

Event Details:

Booth#: 904

Date: November 16-18, 2017

Venue: Calvin L. Rampton Salt Palace Convention Center, Salt Lake City, UT

Website: http://amp17.amp.org/

Poster Presentation:

Poster#: TT76

Category: Technical Topics

Abstract Title: Engineering of Isogenic Cell Lines Using the CRISPR/Cas9 Technology and Precise Characterization of Low Allelic Frequency FFPE Cell Line Blocks for Use as Molecular Reference Standards

Authors: Gianluca Roma, Kevin Yoon, Andrew Hilmer, Sonika Saddar*

* Corresponding author

ABSTRACT:

Introduction:  Cancer is a heterogeneous disease in which hundreds of genes, and many thousands of mutations, have been implicated in oncogenesis.  As we enter an era of precision tumor profiling, there is a significant need for molecular reference standards that can be employed for assay development and quality assurance in order to validate assay performance, and understand cross-site and inter-operator reproducibility.  Cell-line based reference standards are ideal for this application, since they represent a biologically-relevant, reproducible, and renewable source of control materials. With this in mind, we have engineered a reference bank of cell lines that contain over 100 oncogenic point mutations, insertions, and deletions, which are spread across multiple genes, including EGFR, KRAS, BRAF, PIK3CA, NRAS, HRAS, and TP53.  Here, we present data on the utility of these cell lines for reference standard generation, using an engineered, EGFR C797S cell line as a case study.  This mutation is an emerging resistance marker to EGFR T790M-targeted therapeutics.

Methods:  CRISPR/Cas9 targeting reagents were transfected into the RKO cell line, and the pooled, polyclonal population was single-cell cloned by limited dilution.  Positive clones were then identified by Sanger sequencing.  FFPE cell line blocks were prepared in triplicate, and were generated by fixing C797S(+/+) and wild-type cells in neutral-buffered formalin, mixing the cells at pre-defined allelic frequencies (1% and 5% C797S), and embedding the cells in paraffin.  The blocks were then sectioned into FFPE scrolls at 20µm thickness.  In order to assess inter- and intra- block consistency, sections were sampled from the top, middle, and bottom of each block.  For each respective region, 5µm sections were also cut and mounted onto slides for histological analysis by H&E staining.  For genetic analysis, gDNA from FFPE scrolls was extracted, and allelic frequencies were validated by digital PCR (dPCR). 

Results:  Engineered, homozygous C797S clones were conclusively identified by Sanger sequencing.  Derivative FFPE blocks showed a high degree of homogeneity by H&E staining of slide-mounted specimens, with > 70% cell coverage by area.  All scrolls consistently produced >400 ng of total extractable DNA, with intra- and inter- block variabilities of < 10%.  Allelic frequencies were highly reproducible, falling within 10% of the targeted ratio across batches.

Conclusion:  CRISPR/Cas9 is an enabling tool for the generation of precision edited cell lines.  These engineered cell lines can be reproducibly incorporated FFPE-mimetic specimens, and represent an ideal source of genetic reference materials, particularly for difficult to find oncogenic variants.