XLSX file - 607K, Outlier differential expression results in mTNBC for select cancer related genes

XLSX file - 48K, Transcriptome sequencing (RNA-seq) run statistics for mTNBC

XLSX file - 61K, List of somatic point mutations in mTNBC detected by NGS sequencing analysis

PDF file - 135K, Validation of RB1 39bp homozygous deletion in mTNBC1

PDF file - 72K, Profiles of germline/tumor single base mutations by genotype in mTNBC

XLSX file - 598K, edgeR differential expression results in mTNBC for select cancer related genes

PDF file - 1635K, PET/CT images of mTNBC2 breast lesion before and after treatment on combination MEK and AKT inhibitors

XLSX file - 45K, Genes containing breakpoint from translocation in mTNBC

XLSX file - 23K, Mapping statistics for genome sequencing of tumor and germline DNA from mTNBC

XLSX file - 273K, Somatic genic focal copy number changes in mTNBC

XLSX file - 42K, Ingenuity Canonical Pathways results for mTNBC based on edgeR differential expression results

XLSX file - 40K, Characteristics of mTNBC study population

Abstract Triple negative breast cancer (TNBC), is characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). TNBC comprises about 15 of all breast cancer diagnoses, but ∼25% of deaths. TNBC has a more aggressive clinical course and long-term disease-free survival is poor. As is well known, tumors that develop in women who carry deleterious germline BRCA mutations typically have tumors characteristic of the basal-like TNBC phenotype associated with defects in double stand break repair and homologous recombination. These observations have led to the development of therapeutic strategies aimed at these mechanisms, such as PARP inhibitors, which are in mature stages of drug development for the treatment of a subset of patients diagnosed with mTNBC. Iniparib, although not believed to be a true PARP inhibitor, is believed to act through a similar mechanism of action. We have completed a study of whole genome and transcriptome sequencing of 14 patients with metastatic triple negative breast cancer (mTNBC). One patient who was sequenced had a complete response to a combination including gemcitabine, carboplatin, and iniparib prior to sequencing. Sequencing analysis revealed a collection of somatic mutations and alterations in genes associated with DNA repair and homologous recombination, including DCLRE1C, MEI1, TP53, DDB1, RIF1, ZBTB40, BRIP1, and BRCA2. Furthermore, RNA-seq expression analysis demonstrated significant underexpression of BRCA1. Thus, these alterations may serve as markers that can be predictive of responders from non-responders of treatment regimens including iniparib or possibly PARP inhibitors. To begin to identify a genomic profile indicative of PARP response, tumors and blood of four additional mTNBC patient samples were collected and subjected to whole exome and RNA sequencing. We have also obtained the mutational data of TNBC tumors sequenced by the Cancer Genome Atlas. The genetic profiles of these tumors will be compared to the previously sequence mTNBC sample that responded to iniparib to determine the frequency of these specific events in mTNBC. Through this work we hope to identify a genetic profile that can serve as a possible predictor of response to iniparib or true PARP inhibitors. Citation Format: Shukmei Wong, Joyce A. O'Shaughnessy, Patricia LoRusso, Jessica Aldrich, Ahmet Kurdoglu, Alexis Christoforides, Jennifer Dinh, Tyler Izatt, Shripad Sinari, Angela Baker, Tracy M. Moses, Bodour Salhia, Spyro Mousses, Jeffrey Kiefer, Jeffrey M. Trent, Daniel Von Hoff, Geoffrey Shapiro, Winnie Liang, David W. Craig, John D. Carpten. Whole exome and RNA sequencing reveals genetic alterations in DNA damage and homologous recombination pathways in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2013-2023

Abstract Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer. Mol Cancer Ther; 12(1); 104–16. ©2012 AACR.

BACKGROUND:Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers.METHODS:We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations.RESULTS:We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition.CONCLUSIONS:Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source.

Abstract Introduction: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and Her2-neu, and accounts for ∼15% of all breast cancer diagnoses. Adjuvant chremotherapy and surgery can be effective in many women, although relapse with chemoresistant tumors that are difficult to treat is common. Importantly, these tumors may disproportionately affect African American women. Here, we present initial integrated analysis of matched normal and tumor whole genome and tumor transcriptome sequencing data from three African American patients with metastatic chemo-resistant TNBC, to better understand the compendium of somatic events occurring in these tumors within this high-risk population. Methods: Next Generation Sequencing was performed using the SOLiD version 4.0 system. Patient matched tumor and germline genomes are sequenced to 300 million mappable mate-pair reads (30X) and analyzed using custom paired analysis tools to uncover somatic alterations. Tumor transcriptomes are sequenced (RNA-seq) to 30 million uniquely aligned reads using Life Technologies Bioscope. For expression analysis, patient RNA-seq data are compared to data generated from ethnicity-matched population-based control hyperplastic breast tissue using EdgeR and DEGseq. Results: On average, we detected >3 million germline variants in these three African American patients. Review of germline results also provides information on BRCA1/2 status. Somatic paired analysis has revealed point mutations, small indels, copy number alterations, and translocations, in known cancer genes, and has revealed potential novel genes as well. Among three patients sequenced thus far, we have uncovered both unique and common genomic and transcriptomic perturbations. In both patients, unique somatic genomic alterations are associated with upregulation of specific signaling pathways. RNA-seq-based expression analysis has revealed a profile dominated by perturbations in cell cycle mitotic checkpoint defects. Furthermore, integrated analysis has provided key insights into the transcriptional consequences of a number of genomic alterations including exon skipping events and the discovery of potential fusion transcripts. Importantly, DNA and RNA changes have been validated by independent methods and technologies. Conclusions: Deep genome and transcriptome profiling of chemo-resistant TNBC has provided insights into events occurring in this difficult to treat cancer. These data are being leveraged to provide an opportunity for informed therapeutic options for intervention of this difficult to treat form of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2011-LB-267

Our group was the first to implicate the EphB2 tyrosine kinase receptor as a tumor suppressor in prostate cancer (PC), where we reported somatic inactivating mutations occurring in 5%–10% of sporadic tumors. With its role in maintaining normal epithelial architecture and functional data suggestive of a tumor suppressor in both prostate and colon cancer, EphB2 is an attractive candidate genetic risk factor for PC. First, in a screen of EphB2 coding variants in African American familial and sporadic cases and controls, we showed that the frequency of a common germline nonsense mutation (K1019X) was significantly higher in African American men with a positive family history of prostate cancer as compared to matched controls. Following on these findings, we hypothesized that there might be other common variants in EphB2 that confer risk of sporadic prostate cancer among African American men. To test this hypothesis, we genotyped 490 African American cases and 567 matched controls with 335 genetic variants mapping within the EphB2 locus. SNPs genotyped included all haplotype tagging SNPs based on the Yoruban population of Ibadan, Nigeria, International HapMap data, and several novel coding variants discovered by Sanger sequencing. Logistic regression revealed 10 variants showing statistically significant association with prostate cancer risk after correction for multiple testing, including rs4654822 (Odds Ratio = 0.59 (CI=0.47–0.74; P=4.0X10-06). Furthermore, to supplement these data, we used Next Generation Sequencing to interrogate the entire EphB2 genomic locus in a pool of 400 African American cases and controls for discovery of all EphB2 common variants in our cohort. This represents ∼200,000 basepairs of total genomic sequence in 800 haploid genomes or 160,000,000 bases of DNA sequence. Using the Applied Biosystems SOLiD system, we generated >500,000,000 bases of sequence in a single run. We are currently analyzing these data using bioinformatics tools to discover novel common and rare variants in the EphB2 gene, with a focus on regions encompassing SNPs showing association to prostate cancer. We hope to further validate SNPs showing association to prostate cancer and any nearby novel SNPs identified by our sequencing efforts in an independent cohort of men of recent African descent. These data form the basis of an extremely thorough analysis of EphB2 as a genetic risk factor for prostate cancer among men of recent African descent.

Despite its' central role, the precise mechanisms of the phosphoinositide 3-kinase/Akt (PI3K)/Akt pathway activation in acute myeloid leukaemia (AML) have not been elucidated. Recently, a recurrent novel AKT1 pleckstrin homology domain (PHD) mutation leading to membrane translocation, constitutive AKT activation and leukaemia development in mice was described. To assess AKT1 PHD mutations in AML, we sequenced 57 specimens from 49 AML patients, all of whom showed PI3K/AKT pathway activation by analysis of total and phospho-protein expression for AKT, mTor, p70S6Kinase, S6ribosomal protein and PTEN. No mutations in AKT1 PHD were identified, making this mutation an unlikely cause of PI3K/AKT pathway activation in AML.