Chih-Jian Lih

Application of Molecular Profiling in Clinical Trials for Advanced Metastatic Cancers

There is growing interest in the application of molecular profiling, including sequencing, genotyping, and/or mRNA expression profiling, to the analysis of patient tumors with the objective of applying these data to inform therapeutic choices for patients with advanced cancers. Multiple clinical trials that are attempting to validate this personalized or precision medicine approach are in various stages of development and execution. Although preliminary data from some of these efforts have fueled excitement about the value and utility of these studies, their execution has also provoked many questions about the best way to approach complicating factors such as tumor heterogeneity and the choice of which genetic mutations to target. This commentary highlights some of the challenges confronting the clinical application of molecular tumor profiling and the various trial designs being utilized to address these challenges. Randomized trials that rigorously test patient response to molecularly targeted agents assigned based on the presence of a defined set of mutations in putative cancer-driving pathways are required to address some of the current challenges and to identify patients likely to benefit from this approach.

Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

GeneMed: An Informatics Hub for the Coordination of Next-Generation Sequencing Studies that Support Precision Oncology Clinical Trials

We have developed an informatics system, GeneMed, for the National Cancer Institute (NCI) molecular profiling-based assignment of cancer therapy (MPACT) clinical trial (NCT01827384) being conducted in the National Institutes of Health (NIH) Clinical Center. This trial is one of the first to use a randomized design to examine whether assigning treatment based on genomic tumor screening can improve the rate and duration of response in patients with advanced solid tumors. An analytically validated next-generation sequencing (NGS) assay is applied to DNA from patients’ tumors to identify mutations in a panel of genes that are thought likely to affect the utility of targeted therapies available for use in the clinical trial. The patients are randomized to a treatment selected to target a somatic mutation in the tumor or with a control treatment. The GeneMed system streamlines the workflow of the clinical trial and serves as a communications hub among the sequencing lab, the treatment selection team, and clinical personnel. It automates the annotation of the genomic variants identified by sequencing, predicts the functional impact of mutations, identifies the actionable mutations, and facilitates quality control by the molecular characterization lab in the review of variants. The GeneMed system collects baseline information about the patients from the clinic team to determine eligibility for the panel of drugs available. The system performs randomized treatment assignments under the oversight of a supervising treatment selection team and generates a patient report containing detected genomic alterations. NCI is planning to expand the MPACT trial to multiple cancer centers soon. In summary, the GeneMed system has been proven to be an efficient and successful informatics hub for coordinating the reliable application of NGS to precision medicine studies.

Expression array analysis of the hepatocyte growth factor invasive program

Abstract Signaling by human hepatocyte growth factor (hHGF) via its cell surface receptor (MET) drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Oncogenic pathway activation also contributes to tumorigenesis and cancer progression, including tumor angiogenesis and metastasis, in several prevalent malignancies. The HGF gene encodes full-length hHGF and two truncated isoforms known as NK1 and NK2. NK1 induces all three HGF activities at modestly reduced potency, whereas NK2 stimulates only motogenesis and enhances HGF-driven tumor metastasis in transgenic mice. Prior studies have shown that mouse HGF (mHGF) also binds with high affinity to human MET. Here we show that, like NK2, mHGF stimulates cell motility, invasion and spontaneous metastasis of PC3M human prostate adenocarcinoma cells in mice through human MET. To identify target genes and signaling pathways associated with motogenic and metastatic HGF signaling, i.e., the HGF invasive program, gene expression profiling was performed using PC3M cells treated with hHGF, NK2 or mHGF. Results obtained using Ingenuity Pathway Analysis software showed significant overlap with networks and pathways involved in cell movement and metastasis. Interrogating The Cancer Genome Atlas project also identified a subset of 23 gene expression changes in PC3M with a strong tendency for co-occurrence in prostate cancer patients that were associated with significantly decreased disease-free survival.

RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96–0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearmans coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Abstract PL08-02: NCI patient derived models repository

The National Cancer Institute is developing a national repository of patient-derived cancer models (PDMs) comprised of[T] clinically-annotated patient-derived xenografts (PDXs); patient-derived tumor cell cultures (PDCs, including conditionally-reprogrammed tumor cell cultures) prepared from primary and metastatic tumors, circulating tumor cells (CTCs), and/or PDXs; tumor cell lysates, DNA, and RNA; and cancer-associated fibroblast cell lines (CAFs, autologous when possible) to serve as a resource for academic discovery efforts and public-private partnerships for drug discovery. NCI will provide a long-term home for >1000 PDX and PDC models, each produced from tissues and blood supplied by NCI-designated Cancer Centers and NCI-supported clinical trials networks. The effort is targeting the collection of tumors that are less prevalent in current resources, such as: small cell lung cancer, prostate cancer, bladder cancer, pancreatic cancer, head and neck cancers, as well as sarcomas and melanomas. The goals of the project are: (1) to develop a minimum of ∼50 unique patient models (both PDXs and PDCs) per disease such that the size of each molecularly-characterized subgroup is useful for subsequent validation and/or efficacy studies; (2) to perform comprehensive pre-competitive molecular characterization of patient samples and earliest passage PDXs and PDCs that includes the NCI-MPACT mutation panel, WES, RNASeq, copy number determination, histology, growth curves, and pilot proteomic/phospho-proteomic studies; and (3) to make all models and associated pre-clinical and clinical data available through a publicly available website. To date, over 1700 specimens from 1100 patients have been received for the development of PDMs; the overall ‘take9 rate for PDXs originating from solid tumors is 70% with >170 assessable models and another 270 early passage tumors currently in evaluation. As expected, based on collection priorities, tumors of genitourinary, digestive, head and neck, musculoskeletal, respiratory, and skin origin are the major histological sites of origin for our PDX models. In addition, over 90 conditionally-reprogrammed cell lines have been expanded from both 18-gauge needle biopsies and surgical resections, and have passed initial quality control procedures; many of these cell cultures have a matched PDX. Over 150 CAF lines have been developed following repeated (>10) purification steps using flow cytometry and are in the process of quality control procedures that demonstrate complete lack of growth in NOD-SCID gamma IL2 receptor null (NSG) mice; of these CAFs, we have developed matched pairs of PDCs and CAFs from the same patient in 16 cases. To evaluate the potential utility of the NCI PDM Repository, we have prospectively ‘entered9 22 models in a pre-clinical trial for which eligibility (based on actionable mutations) and treatment arms are identical to those in the NCI-MPACT study (NCT01827384). Multiple objective responses (significant improvement in overall survival) have been observed in all arms of the study and in a variety of models. WES and RNASeq analysis have proven essential to explain the therapeutic responses that we have observed. We are also evaluating the relationship of in vitro and in vivo activity for the NCI-MPACT drug panel in the models where concurrent PDCs and PDXs have been produced. A web site has been developed that will provide annotated information on the models (such as DNA sequence, gene expression, prior therapy) to investigators to assist in the distribution of the contents of the repository to the research community. We expect to be able to begin distribution in late spring of 2016. Citation Format: James H. Doroshow, Melinda Hollingshead, Yvonne Evrard, P. Mickey Williams, Vivekananda Datta, Biswajit Das, Chih-Jian Lih, Dianne Newton. NCI patient derived models repository. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL08-02.

Moving next-generation sequencing into the clinical realm: Detection of somatic mutations in cancer by targeted amplicon sequencing.

60 Background: Molecular targeted therapies are increasingly important in treating cancer patients; robust analytically validated clinical assays are required for patient selection in early-stage clinical trials. The goal of Molecular Characterization Laboratory (MoCha) is to develop clinical diagnostic assays using next generation sequencing methods to support clinical studies in DCTD (CTEP). METHODS We developed a custom assay for somatic mutation detection using Fluidigm access array technology for amplicon generation followed by sequencing with the Illumina Miseq. A panel of 62 amplicons covering 6 Kb genomic regions was designed to detect 92 DNA loci, including common therapeutically actionable targets, in 37 genes. Analytical studies were performed using genomic DNA samples from fresh or formalin fixed cancer cell-lines and a normal hapmap individual (CEPH). We subsequently applied this assay to characterize DNA samples from both tumor tissues and blood specimens from ovarian cancer patients. RESULTS The assay detected known variants in both frozen and fixed DNA samples reproducibly with high sensitivity and specificity (<2%). Using a series of positive control plasmid spikes mixed into a normal reference CEPH DNA at pre-defined copy number ratios, we verified the assay is sensitive to detect variants at 5% allelic frequency with a minimum 400 X coverage. We identified somatic mutations in TP53 and PIK3CA in a few patients, and a germ-line variant D1583N in ATM genes occurring in one-third of tested patients. CONCLUSIONS We developed and validated a next generation sequencing assay suitable for patient selection for clinical trials. Plans are to correlate sequencing and clinical results when clinical data are available.