Abstract: PNFBA-12
Sources of Funding: Supported by:_x000D_ The John and Mary Brock Foundation_x000D_ The Claude P. Cook Foundation_x000D_ Veterans Affairs Merit award to Dr. Petros_x000D_ The Coca?Cola Company Research Award_x000D_ Philanthropic award given by the Emory University Winship Cancer Institute

Introduction

Circulating tumor DNA (ctDNA) has characteristics of an ideal biomarker. We sought to develop whole exome sequencing of ctDNA to interrogate commonly mutated genes in renal cell carcinoma for early tumor detection through a single blood sample.

Methods

Patients with solid renal tumors and healthy controls gave 40 mL blood and plasma cell free DNA was prepared. A multiplex bar coded polymerase chain reaction amplification using the Fluidigm Access Array was performed to prepare sequencing libraries for the Illumina HiSeq platform. Galaxy workflow was used to identify mutations and results were compared to buffy coat sequencing. The following genes were queried: VHL, PBRM1, SETD2, BAP1, KDM5C, KIT, NFE2L2, MET, TP53, CDKN2A, FGFR3, PIK3CA, BRAF, MUC4. Criteria for calling mutations included adequate frequency by overall count and percentage of reads, identification in all overlapping sequences, and presence of buffy coat for comparison with <0.5% containing the mutation.

Results

Thirty preoperative test patients with RCC and 32 healthy controls were analyzed using the gene panel. Of the 32 patients analyzed in the healthy control cohort, 27 (84%) failed to yield sequence of the genes of interest. Of the pre operative RCC patients, 20/30 (67%) had detectable somatic mutations, resulting in nonsynonymous, frameshift, stopgain, or splice site mutations, compared to 1/32 (3.1%) controls. Mutations were detected in both early and advanced stage disease, including a patient with a 1.1 x 0.7 x 0.5 cm tumor. Mutations were seen in all genes assayed.

Conclusions

These data demonstrate feasibility of gene-specific whole exome sequencing of ctDNA for diagnosis of RCC in patients with solid renal tumors. The majority of RCC patients of various stages and histology had ctDNA detected in a single preoperative blood sample. A single control gave a positive test. Non invasive detection of RCC shows promise for not only initial diagnosis but also disease monitoring and guidance of targeted therapies throughout a wide spectrum of disease severity, including small lesions.

Funding

Supported by:_x000D_ The John and Mary Brock Foundation_x000D_ The Claude P. Cook Foundation_x000D_ Veterans Affairs Merit award to Dr. Petros_x000D_ The Coca?Cola Company Research Award_x000D_ Philanthropic award given by the Emory University Winship Cancer Institute