Abstract 615: Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma
Authors: Kaedbey Rayan , Kis Olena , Danesh Arnavaz , Dowar Mark , Li Tiantian , Li Zhihua , Liu Jessica , Mansour Mark , Sukhai Mahadeo , Zhang Tong , Kamel-Reid Suzanne , Pugh Trevor J. , Trudel Suzanne 
Published: August 2015

Abstract
Background: Genome sequencing of multiple myeloma (MM) tumors has revealed recurrent mutations that serve as a fertile ground for targeted therapies. Indeed, activating mutations of KRAS, NRAS and BRAF have been reported in approximately 27%, 24% and 4% of MM cases. Based on this observation, we initiated a Phase II NCI-CTEP sponsored clinical trial of trametinib in patients with MM (PHL-9460). Detection of mutations currently require bone marrow aspirates that are invasive and can yield suboptimal samples. Targeted, ultra-deep sequencing of circulating tumor DNA (ctDNA) is a promising tool for accessing the tumor genome that has not been well studied in MM. We set out to determine the feasibility of detecting ctDNA in MM and of identifying actionable mutations and mutational load using liquid biopsy through ctDNA analysis.
Methods: MM patients enrolled onto PHL-9460 or those with heavy tumor burden were identified and consented to have their peripheral blood drawn for analysis. Where possible, matched tumor DNA was also obtained. Cell-free DNA was extracted from 7-15 mL of plasma isolated within ¬1 hour of blood draw using the QIAamp Circulating Nucleic Acid Kit and tagged with barcoded sequencing adapters for subsequent pooling. All exons of KRAS, NRAS, BRAF, PIK3CA and EGFR genes were isolated using a custom hybrid capture panel (IDT xGen Lockdown) and sequenced on an Illumina HiSeq 2000. Reads were aligned to the human genome reference (hg19) using bwa and somatic mutations were detected using muTect.
Results: We have collected 25 samples from 23 patients, 7 from patients on PHL-9460, 5 newly diagnosed, and 13 from relapsed patients having received 3.3 median prior lines of therapy (range 1-7). To date, 11 samples from 10 patients have been sequenced. The sample with the lowest DNA yield failed due to low library complexity (range 16.6-3872 ng, median yield: 197 ng). From the remaining 10 samples, the mean target coverage ranged from 31,500 to 32,500. Somatic mutations in KRAS, NRAS, or PIK3CA genes were present in 5 of 9 patients with mutant allele frequencies ranging from 1.1% to 32% (3 KRAS and 2 NRAS of which 2 cases also had a low frequency PIK3CA mutation). We did not uncover mutations in BRAF or EGFR. For patients with matched tumor DNA, mutations in ctDNA concurred with those found in tumor DNA sequencing (4 of 4 tumors with known genotypes). Two patients with NRAS mutations enrolled onto PHL-9460 have responded to trametinib (1 partial and 1 minor response) and remain on therapy.
Conclusion: ctDNA analysis in this cohort has identified key mutations in MM. The rate of RAS or RAF mutations in ctDNA compared to matched tumors and correlation to response to trametinib is ongoing and will be presented. Preliminary data suggest that ctDNA may be a reliable method of detecting mutations in MM and an alternative to bone marrow biopsy.
Citation Format: Rayan Kaedbey, Olena Kis, Arnavaz Danesh, Mark Dowar, Tiantian Li, Zhihua Li, Jessica Liu, Mark Mansour, Mahadeo Sukhai, Tong Zhang, Suzanne Kamel-Reid, Trevor J. Pugh, Suzanne Trudel. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 615. doi:10.1158/1538-7445.AM2015-615