Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.
Abstract Background Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer‐associated genes as an efficient approach to characterizing patient samples (compared to whole‐exome and whole‐genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost‐effective method for genomic assessment of patient‐derived cell lines to evaluate fidelity to initial patient tumor samples. Methods We developed a prostate cancer‐specific targeted next‐generation sequencing (NGS) panel to detect alterations in 62 prostate cancer‐associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient‐derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. Results The prostate cancer‐specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell‐free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient‐derived cell lines; however, no prostate cancer‐specific mutations were identified in the tumor‐derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. Conclusions We evaluated a prostate cancer‐specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh‐frozen tumors, cell‐free DNA, CTCs, and cell lines. Targeted sequencing of patient‐derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer‐specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.
11513 Background: The ability to perform multiple genomic tests on metastatic (met) bx may enable cancer research and precision medicine. Questions remain about feasibility of obtaining multiple met cores, usability of met bx for genomics, and utility of genomic profiling for identifying relevant targets. Methods: We developed a prospective bx protocol where multiple research bx are collected from mets and used for: pathology; ER/PR/HER2; OncoPanel, a CLIA 300 gene sequencing (seq) panel; whole exome seq (WES); transcriptome seq (RNA-seq); and single-cell RNA-seq (sc-RNA-seq). In some cases, tissue is used for cell line and xenograft generation. Archival primary bx is obtained when possible for parallel genomic studies. Patients (pts) are followed and serial bx obtained. Cell free DNA (cfDNA) is collected at time of biopsy and serially. Results: We obtained 51 met bx from 49 pts. 6 cores were taken per bx on avg (range 1-12). The number of bx with sufficient material for each test and success rates is shown in the Table. cfDNA was collected at 88 timepoints from 46 pts. In 2/51 bx, pathology found a tumor type other than breast cancer. Of the remaining 49, 12% changed ER or HER2 status. Of pts w OncoPanel data, 37.5% had ESR1 LBD mutations, 50% PIK3CA mutations, 16% ERBB2 mutations, and 3% FGFR1 amplifications. Overall, 75% had alterations in at least one of these genes, which are criteria for trials at our institution. Many pts had alterations in genes involved in resistance to therapies in preclinical studies (RB1, MYC, CCND1, NF1). Conclusions: A met bx program for cancer precision medicine is feasible, with ~90% of bx yielding sufficient tissue for pathology, receptors, CLIA targeted sequencing, WES, and RNAseq. Met bx have clinical implications, identifying a different cancer (4%), different targetable receptors (12%), and specific trials (>75%). For many genes, the mutational landscape differs significantly from primary breast cancer—highlighting the value of met bx. Sufficient to Test Completed Pending Successful Pathology/Receptors 51 (100%) 51 0 51 (100%) OncoPanel (CLIA) 49 (96%) 38 11 35 (92%) WES 45 (88%) 29 16 28 (97%) RNA-seq 45 (88%) 14 31 12 (86%) sc-RNA-seq 38 (75%) 1 37 1 (100%)
<p>Supplemental Figure 1. Copy number alterations of AAPC exome set. Supplemental Figure 2. Copy number analysis grouped by Gleason scores. Supplemental Figure 3. Recurrent somatic copy number alterations in the AAPC cohort. Supplemental Figure 4. Analysis of fraction of copy number altered genome in the AAPC exome and TCGA cohorts. Supplemental Figure 5. Prostatic adenocarcinomas with unique ERF mutations demonstrate decreased RNA expression by RNA ISH. Supplemental Figure 6. IGV screenshots of validated ERF mutations. Supplemental Figure 7. Frequencies of mutations in ERF across several published prostate cancer sequencing cohorts. Supplemental Figure 8. Visualization of deletions at chr19q13.2 in the TCGA cohort and analysis of SU2C/CRPC dataset for ERF mutations. Supplemental Figure 9. Association ERF deletion status with pathologic features. Supplemental Figure 10. Knockdown of ERF mRNA in prostate cancer cell lines and an immortalized prostate epithelial cell line. Supplemental Figure 11. Knockdown of ERF in PC�3 cell line augments invasion and tumor xenograft growth. Supplemental Figure 12. Overexpression of ERF in PC�3 cell line is associated with a growth inhibitory effect. Supplemental Figure 13. Knockdown of ERF in RWPE�1 and LNCaP cell lines contributes to growth proliferation. Supplemental Figure 14. Overexpression of ERF in DU�145 cell line. Supplemental Figure 15. Association of LHS�AR ERF KD signature in the CCLE and CRPC datasets. Supplemental Figure 16. ERF mutation and deletions are mutually exclusive from ERG rearrangement events in the published TCGA cohort (n=333) (cbioportal.org). Supplemental Figure 17. ERF KD signature in setting of ERF overexpression and correlation with Gleason score. Supplemental Figure 18. Association of LHS�AR ERF KD signature projected across the CCLE. Supplemental Figure 19. Mutation plot of combined AAPC (n=102) + TCGA (n=457) analysis. Supplemental Figure 20. ERF nucleotide sequence map.</p>
African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (
Abstract Background: While great strides have been made in the treatment of estrogen receptor-positive (ER+) metastatic breast cancer (MBC), therapeutic resistance invariably occurs. A better understanding of the underlying resistance mechanisms is critical to enable durable control of this disease. Methods: We performed whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) on metastatic tumor biopsies from 88 patients with ER+ MBC who had developed resistance to one or more ER-directed therapies. For 27 of these patients, we sequenced the treatment-naïve primary tumors for comparison to the resistant specimens. Tumors were analyzed for point mutations, insertions/deletions, copy number alterations, translocations, and gene expression. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of all metastatic samples demonstrated several recurrently altered genes whose incidence differed significantly from primary, treatment-naïve ER+ breast cancers sequenced in the TCGA study (TCGA). These include ESR1 mutations (n=17, 19.3%; 32.86 fold enrichment, q.value<7.5e-12), CCND1 amplification (n=52, 59.1%; 2.3 fold enrichment, q.value<0.0073), and MAP2K4 biallelic inactivation (n=14, 15.9%; 3.04 fold enrichment, q.value< 0.054). Comparing to matched primary samples from the same patient, many alterations were found to be acquired in several cases, including for ESR1, ERBB2, PIK3CA, PTEN, RB1, AKT1, and others. Initial analysis of RNA-seq data from metastatic samples (n=59) allowed classification of individual resistance mechanisms into broader resistance modes based on the observed transcriptional state. Conclusions: We present a genomic landscape of resistant ER+ MBC using WES and RNA-seq. Multiple genes were recurrently altered in these tumors at significantly higher rates than in ER+ primary breast cancer. When compared with matched primary tumors from the same patient, alterations in these and other genes were often found to be acquired after treatment, suggesting a role in resistance to ER-directed therapies and/or metastasis. Potential resistance mechanisms appear to fall into several categories; integrating RNA-seq data may enhance the ability to identify these categories even when genomic alterations are not identified. Multiple clinically relevant genomic and molecular alterations are identified in metastatic biopsies– with implications for choice of next therapy, clinical trial eligibility, and novel drug targets. Citation Format: Cohen O, Kim D, Oh C, Waks A, Oliver N, Helvie K, Marini L, Rotem A, Lloyd M, Stover D, Adalsteinsson V, Freeman S, Ha G, Cibulskis C, Anderka K, Tamayo P, Johannessen C, Krop I, Garraway L, Winer E, Lin N, Wagle N. Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-01.
