Abstract 06: Comparing mutational profiles between cell-free circulating tumor DNA and tumor DNA in laryngeal carcinoma patients
Authors: McMullen Hunter , Greer Adam , Khandelwal Alok R. , Mickie Hamiter , Ma Xiaohui , Moore-Medlin Tara , Hong Yin , Mills Glenn , Nathan Cherie-Ann O. 
Published: December 2017

Abstract
Introduction: The incidence of laryngeal cancers is on the rise with an estimated 12,000 new cases diagnosed each year in the United States. Despite advancements in surgery, chemotherapy, radiotherapy, and targeted therapy, the prognosis for advanced laryngeal cancer patients is poor due to local invasion and metastasis. Identifying biomarkers that can predict response to therapy and potential recurrence in patients is critical to improving survival. Cell-free, circulating tumor DNA (ctDNA) can act as a noninvasive cancer biomarker, offering a potential alternative to invasive tissue biopsies for recurrent disease. This technology can be used to monitor tumor progression and metastasis and potentially guide therapy.
Objective: Using next-generation sequencing, we aimed to determine whether the ctDNA mutational profile could potentially predict recurrence in advanced stage laryngeal carcinoma patients.
Methods: Genomic DNA was isolated from primary laryngeal tumors using a NucleoSpin Tissue kit by Clontech. Matched circulating cell-free DNA (cfDNA) was extracted from plasma samples of the same patient using Zymo's Quick-cfDNA™ Serum & Plasma Kit. DNA concentrations were measured using Qubit 2.0. The quality and size of cfDNA was determined using Agilent Tapestation 2200. Libraries were prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences. The pair-end DNA sequencing was conducted on Illumina's MiSeq platform with MiSeq Reagent Kits v2 (2x150). Variants were identified by Biomedical Genomic Workbench (Qiagen) according to quality parameters. The cutoff value for allele frequency of variants in tissue DNA and ctDNA was 5% and 0.5%, respectively.
Results: Total of 12 paired laryngeal cancer tissue and cfDNA samples were sequenced. Four pairs were from non-recurrence patients and eight were from patients with recurrent disease. The DNA sequencing results showed shared germline non-synonymous variants between tissue and cfDNA samples in three of the four non-recurrent patients. The variants occurred in gene TP53, KDR, ATM, and ERBB4. The allele frequency of variants was similar in each pair of DNA samples. Depending on a homozygous or heterozygous variant, the allele frequency was about 100% and 50% respectively. In one non-recurrent patient, matched non-synonymous somatic variants in both the tumor tissue and cfDNA were detected. The mutant genes and allele frequency of variants (tissue vs cfDNA in %) were PIK3CA (32.4 vs 2.8) G106C, TP53 (26.6 vs 2.2) R249del, TP53 (36.4 vs 4.5) S90fs. In paired recurrent laryngeal samples, non-synonymous somatic variants determined in tissue DNA were detected in cfDNA from 3 of 8 patients. These variant genes were TP53 (30.7 vs 1.4) P190T, HNF1A (6.4 vs 1.4) M283fs, HNF1A (10.1 vs 2.6) M283I, TP53 (58.1 vs 1.0) E192*, and TP53 (8.5 vs 1.1) P72R. In addition, some somatic variants with allele frequency over 5% were found only in cfDNA, but not found in tissue DNA in 6 recurrent patients. These variant genes were ATM (I173fs, L1708P, M1714fs), KIT (F689fs, S692fs), HNF1A (W219*), TP53 (I225N), and MPL (S505N). The somatic mutation in genes could have deleterious effects in DNA repair, cell growth and regulation, gene transcription, and tumor suppression. Our finding suggests that non-synonymous somatic variants found in cfDNA are ctDNA. Further, the somatic variants found only in cfDNA but not in tissue DNA suggest there may be a small portion of tumor cells with a high apoptotic rate in heterozygous cancer tissue of recurrent patients. These variants in cfDNA could be indicators of tumor recurrence.
Conclusion: CfDNA from recurrent patient samples showed a high detection rate of somatic mutations compared to non-recurrent patient samples. Our study supports the development of ctDNA mutational profiling as a predictive biomarker for recurrent laryngeal carcinoma.
Citation Format: Hunter McMullen, Adam Greer, Alok R. Khandelwal, Hamiter Mickie, Xiaohui Ma, Tara Moore-Medlin, Yin Hong, Glenn Mills, Cherie-Ann O. Nathan. Comparing mutational profiles between cell-free circulating tumor DNA and tumor DNA in laryngeal carcinoma patients [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 06.