Abstract 3509: Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry.
Authors: Pelak Kimberly , Wright Jennifer , Yan Zhenyu , Darwanto Agus , Liu Weihua , Fang Peng , Li Jin , Sankar Sabita , Galderisi Chad 
Published: August 2014

Abstract
Introduction: LKB1 (liver kinase B1)/STK11 (serine-threonine kinase 11) is a tumor suppressor that encodes a serine/threonine kinase which negatively regulates the mTOR (mammalian target of rapamycin) signaling pathway. Somatic mutations in LKB1 occur most notably in lung adenocarcinoma, cervical cancers and melanoma. Inactivation of LKB1 is caused by point mutations, homozygous deletions or promoter methylation. LKB1 mutation status is a predictive marker for responsiveness to both MEK and PI3K inhibitors. We have developed next-generation sequencing (NGS), Sanger sequencing and immunohistochemistry (IHC) assays to assess LKB1 mutation and protein expression status in a set of FFPE patient specimens.
Methods: For NGS, we used the Ion Torrent platform to sequence a 4.4kb region of interest (ROI) around LKB1. A combination of TorrentSuite and in-house tools were used for alignment, variant calling and prioritization of variants. Sanger sequencing was used to validate the presence and absence of variants with a frequency of at least 10% that were identified by NGS. An IHC assay was developed using a commercially available antibody. These assays were validated using a combination of cell lines, a genetically engineered mouse model and FFPE tissue for specificity, sensitivity, reproducibility and concordance.
Results: 35 patient FFPE tissues from a variety of tumors including cervix, endometrium, pancreas, skin and lung were evaluated for LKB1 mutation status using NGS and IHC methods. About 96% of the coding region of LKB1 had sufficient sequencing coverage (read depth &gt;500x) to reliably call low frequency somatic variants. Analytical validation using cell line dilutions demonstrated that our NGS assay is able to detect single base changes with frequencies as low as 2-5%, and small indels with frequencies as low as 5-10%.
We identified 9 coding and splice site variants in our preliminary analysis of 18/35 of the FFPE specimens. We compared the variants in 7 of these FFPE samples to the variants identified in these samples by Sanger sequencing, and found 100% concordance between the variant calls, for NGS variants with frequencies greater than 20%. Sanger sequencing in the remaining FFPE samples is ongoing, and lower frequency NGS variants will be confirmed by other methods. We compared the validated NGS variants with the IHC results to examine the correlation of sequencing results with protein expression analysis. In our preliminary analysis of the first 18 FFPE samples, all of the samples with an IHC score of zero or one (IHC negative) had either a nonsense (n=1) or missense (n=4) variant in STK11.
Conclusions: Our data suggest that accurate assessment of LKB1 status may require complementary methods including NGS and IHC methods.
Citation Format: Kimberly Pelak, Jennifer Wright, Zhenyu Yan, Agus Darwanto, Weihua Liu, Peng Fang, Jin Li, Sabita Sankar, Chad Galderisi. Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry. [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 3509. doi:10.1158/1538-7445.AM2013-3509