James Gao

A specific missense mutation in GTF2I occurs at high frequency in thymic epithelial tumors

We analyzed 28 thymic epithelial tumors (TETs) using next-generation sequencing and identified a missense mutation (chromosome 7 c.74146970T>A) in GTF2I at high frequency in type A thymomas, a relatively indolent subtype. In a series of 274 TETs, we detected the GTF2I mutation in 82% of type A and 74% of type AB thymomas but rarely in the aggressive subtypes, where recurrent mutations of known cancer genes have been identified. Therefore, GTF2I mutation correlated with better survival. GTF2I β and δ isoforms were expressed in TETs, and both mutant isoforms were able to stimulate cell proliferation in vitro. Thymic carcinomas carried a higher number of mutations than thymomas (average of 43.5 and 18.4, respectively). Notably, we identified recurrent mutations of known cancer genes, including TP53, CYLD, CDKN2A, BAP1 and PBRM1, in thymic carcinomas. These findings will complement the diagnostic assessment of these tumors and also facilitate development of a molecular classification and assessment of prognosis and treatment strategies.

Whole Genome and Transcriptome Sequencing of a B3 Thymoma

Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomic hybridization using Agilent platform, transcriptome sequencing using HiSeq 2000 (Illumina) and whole genome sequencing using Complete Genomics Inc platform. Whole genome sequencing determined, in tumor and normal, the sequence of both alleles in more than 95% of the reference genome (NCBI Build 37). Copy number (CN) aberrations were comparable with those previously described for B3 thymomas, with CN gain of chromosome 1q, 5, 7 and X and CN loss of 3p, 6, 11q42.2-qter and q13. One translocation t(11;X) was identified by whole genome sequencing and confirmed by PCR and Sanger sequencing. Ten single nucleotide variations (SNVs) and 2 insertion/deletions (INDELs) were identified; these mutations resulted in non-synonymous amino acid changes or affected splicing sites. The lack of common cancer-associated mutations in this patient suggests that thymomas may evolve through mechanisms distinctive from other tumor types, and supports the rationale for additional high-throughput sequencing screens to better understand the somatic genetic architecture of thymoma.

Abstract 366: A novel transcript variant of androgen receptor identified in circulating tumor cells from castration-resistant prostate cancer patients as a potentially prognostic biomarker

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Circulating tumor cells (CTCs) are rare cancer cells in the bloodstream thought to have a key role in cancer metastasis. There are enormous interests in their analysis for castration-resistant prostate cancer (CRPC) due to their tendency to metastasize to bone. In this study, we applied a surface marker-independent microfluidic device for CTCs capture and retrieval that is based on cell size and deformability. Our device was able to capture 90% and recover 30% of tumor cells in spike experiments. We also developed and validated assays capable of detecting the transcript levels of selected molecular markers in single cells prepared with C1TM Single-Cell AutoPrep System. Blood samples drawn from 34 CRPC patients and 10 primary prostate cancer patients (PPC) were preceded for CTC isolation and molecular characterization. We found that 73% CRPC patients and 10% PPC patients were positive for EpCAM, and only about half of the EpCAM-positive CRPC cases were PSA or AR positive. It is interesting that while there is a strong association between AR and PSA expression in CTC samples, there is no correlation between AR or PSA transcripts in CTCs and serum PSA protein levels in CRPC patients. The positive detection of AR or PSA, but not EpCAM is statistically associated with poor prognosis of the CRPC patients. Very intriguingly, we identified a novel androgen receptor (AR) transcript variant -ARv from four CRPC patients’ CTCs. This ARv lacks a 554bp sequence region in the ligand binding domain (LBD), resulting in a loss of the entire LBD. Clinical data analysis revealed that the ARv is significantly associated with worse survival of CRPC patients (p<0.0001). In vitro functional studies showed this novel ARv is constitutively active in the absence of testosterone. Our findings suggest that this AR variant might contribute to the late stage progression of the diseases and could be used as a potential indicator for alternative therapies. In summary, our new fluidic device is capable of isolating CTC to enable molecular marker analysis, providing value in prognosis and guidance to therapy. Citation Format: Zhigang Kang, Avani Shah, Yunkai Yu, Yuelin Zhu, Ali Asgar Bhagat, Kyra Zhao, Andrew Wu, James Gao, Ravi Madan, James Gulley, William Dahut, Paul Meltzer, Liang Cao. A novel transcript variant of androgen receptor identified in circulating tumor cells from castration-resistant prostate cancer patients as a potentially prognostic biomarker. [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 366. doi:10.1158/1538-7445.AM2015-366